Polypeptides comprising immunoglobulin single variable domains targeting il-13 and tslp

ABSTRACT

The present technology aims at providing a novel type of drug for treating a subject suffering from an inflammatory disease. Specifically, the present technology provides polypeptides comprising at least four immunoglobulin single variable domains (ISVDs), characterized in that at least two ISVDs bind to IL-13 and at least two ISVDs binds to TSLP. The present technology also provides nucleic acids, vectors and compositions.

RELATED APPLICATIONS

This application is a division of U.S. application Ser. No. 17/208,046,filed Mar. 22, 2021, which is a continuation of U.S. application Ser.No. 17/115,906, filed Dec. 9, 2020, which claims the benefit under 35U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 62/945,391,filed Dec. 9, 2019, the entire contents of each of which areincorporated by reference herein in its entirety.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Nov. 15, 2021, isnamed A0848.70215US05-SEQ-JRV, and is 99,569 bytes in size.

1. FIELD OF THE PRESENT TECHNOLOGY

The present technology relates to polypeptides targeting IL-13 and TSLP.It also relates to nucleic acid molecules encoding the polypeptide andvectors comprising the nucleic acids, and to compositions comprising thepolypeptide, nucleic acid or vector. The present technology furtherrelates to these products for use in a method of treating a subjectsuffering from an inflammatory disease. Moreover, the present technologyrelates to method of producing these products.

2. TECHNOLOGICAL BACKGROUND

While necessary for host-defense, unrestrained immune responses can leadto a range of inflammatory diseases such as asthma, atopic dermatitisand rheumatoid arthritis. A cascade of immune responses mediated by theinnate and adaptive arms of the immune system (e.g., antigenrecognition, antigen processing, antigen presentation, cytokineproduction, antibody production, target cell killing) drive theinitiation and propagation of a range of immunological diseases.Inflammatory diseases are often chronic and can even belife-threatening. Allergic and atopic diseases such as asthma and atopicdermatitis are driven predominantly by type 2 immune responses andcharacterized by salient features of type 2 immunity such as high IgEproduction and eosinophilia.

Thymic stromal lymphopoietin (TSLP) and Interleukin-13 (IL-13) aresoluble cytokine targets produced by stromal and/or immune cells(Ziegler & Artis, Nat Rev Immunol (2010) 11:289, Gieseck III et al., NatRev Immunol (2018) 18:62). Human TSLP and IL-13 drive distinct,overlapping and synergistic aspects of type 2 immunity, type 2inflammatory diseases such as asthma and atopic dermatitis as well as abroad array of immunological diseases.

The signalling of TSLP begins through a heterodimeric receptor complexcomposed of the thymic stromal lymphopoietin receptor (TSLPR) and theIL-7R alpha chain (IL-7Rα). Similarly, IL-13 signalling starts bybinding to a heterodimeric receptor complex consisting of alpha IL-4receptor (IL-4Rα) and alpha Interleukin-13 receptor (IL-13R1α). The highaffinity of IL-13 to the IL-13R1 leads to their complex formation whichfurther increase the probability of a heterodimer formation to IL-4Rα.

TSLP drives the maturation of dendritic cells, development andproliferation of mast cells, as well as activating other immune cellssuch as basophils and innate lymphoid cells (ILC2). Similarly, IL-13exerts a range of immunopathologies such as epithelial barrierdisruption, mucus production from mucosal-epithelial surfaces, airwayremodeling as well as the induction of eosinophil recruiting chemokinessuch as eotaxin. These mechanisms are central to the initiation andpropagation of the type 2 inflammatory response and are central to thedevelopment of a range of immunopathologies in diseases such as atopicdermatitis and asthma.

Currently, patients with moderate/severe asthma are inadequatelyresponding to presently available standard of care treatments, includingbiological such as anti-IL4Rα monoclonal antibody Dupixent (dupilumab;marketed), anti-IL5s (marketed), anti-IgE monoclonal antibody Xolair(omalizumab; marketed), in particular in asthma patients with alow-eosinophilic phenotype. Although antagonistic monoclonal antibodiesagainst TSLP (tezepelumab) and IL-13 (lebrikizumab) are presentlyundergoing clinical trials, there are no active clinical developmentprograms that target both TSLP and IL-13. Dual targeting of TSLP andIL-13 with a single agent has the potential to confer efficacy in bothlow-type 2 and high-type 2 asthma as well as atopic dermatitis, with thepotential to confer efficacy in sub-populations within these indicationswhere a single monospecific agent therapy may not be fully efficacious.Accordingly, there is still an unmet medical need for the treatment oftype 2 inflammatory diseases such as asthma and atopic dermatitis thatis not only more efficacious but also conveniently applicable to thepatient.

Such therapy may comprise targeting multiple disease factors, such asIL-13 and TSLP.

Targeting multiple disease factors may be achieved for example byco-administration or combinatorial use of two separate biologicals, e.g.antibodies binding to different therapeutic targets. However,co-administration or combinatorial use of separate biologicals can bechallenging, both from a practical and a commercial point of view. Forexample, two injections of separate products result in a moreinconvenient and more painful treatment regime to the patients which maynegatively affect compliance. With regard to a single injection of twoseparate products, it can be difficult or impossible to provideformulations that allow for acceptable viscosity at the requiredconcentrations and suitable stability of both products. Additionally,co-administration and co-formulation requires production of two separatedrugs which can increase overall costs.

Bispecific antibodies that are able to bind to two different antigenshave been suggested as one strategy for addressing such limitationsassociated with co-administration or combinatorial use of separatebiologicals, such as antibodies.

Bispecific antibody constructs have been proposed in multiple formats.For example, bispecific antibody formats may involve the chemicalconjugation of two antibodies or fragments thereof (Brennan, M, et al.,Science, 1985. 229(4708): p. 81-83; Glennie, M. J., et al., J Immunol,1987. 139(7): p. 2367-2375).

Disadvantages of such bispecific antibody formats include, however, highviscosity at high concentration, making e.g. subcutaneous administrationchallenging, and in that each binding unit requires the interaction oftwo variable domains for specific and high affinity binding, comprisingimplications on polypeptide stability and efficiency of production. Suchbispecific antibody formats may also potentially lead to Chemistry,Manufacturing and Control (CMC) issues related to mispairing of thelight chains or mispairing of the heavy chains.

3. SUMMARY OF THE PRESENT TECHNOLOGY

In some embodiments, the present technology relates to a polypeptidetargeting specifically IL-13 and TSLP at the same time leading to anincreased efficiency of modulating a type 2 inflammatory response ascompared to monospecific anti-IL-13 or anti-TSLP polypeptides in vitro.In some embodiments, the polypeptides are efficiently produced (e.g. inmicrobial hosts). Furthermore, in some embodiments, such polypeptideshave limited reactivity to pre-existing antibodies in the subject to betreated (i.e., antibodies present in the subject before the firsttreatment with the antibody construct). In other embodiments suchpolypeptides exhibit a half-life in the subject to be treated that islong enough such that consecutive treatments can be conveniently spacedapart.

In one embodiment, the present technology provides a polypeptidecomprising or consisting of at least one immunoglobulin single variabledomain (ISVD) that specifically binds to IL-13. In a further embodiment,the polypeptide of the present technology comprises or consists of atleast two ISVDs that specifically bind to IL-13, wherein the two ISVDsare optionally linked via a peptidic linker. In one embodiment, the twoISVDs specifically binding to IL-13 are distinct ISVDs. Moreover, in oneembodiment the polypeptide further comprises one or more other groups,residues, moieties or binding units, optionally linked via one or morepeptidic linkers, in which said one or more other groups, residues,moieties or binding units provide the polypeptide with increasedhalf-life, compared to the corresponding polypeptide without said one ormore other groups, residues, moieties or binding units. For example, thebinding unit can be an ISVD that binds to a (human) serum protein, suchas human serum albumin.

In another aspect, the polypeptide of the present technology comprisesor consists of at least one ISVD that specifically binds to TSLP. In afurther embodiment, the polypeptide of the present technology comprisesor consists of at least two ISVDs that specifically bind to TSLP,wherein the two ISVDs are optionally linked via a peptidic linker. Inone embodiment, the two ISVDs specifically binding to TSLP are distinctISVDs. Moreover, in one embodiment the polypeptide further comprises oneor more other groups, residues, moieties or binding units, optionallylinked via one or more peptidic linkers, in which said one or more othergroups, residues, moieties or binding units provide the polypeptide withincreased half-life, compared to the corresponding polypeptide withoutsaid one or more other groups, residues, moieties or binding units. Forexample, the binding unit can be an ISVD that binds to a (human) serumprotein, such as human serum albumin.

In another embodiment, the polypeptide of the present technologycomprises or consists of at least four ISVDs, wherein at least two ISVDsspecifically bind to IL-13 and at least two ISVDs specifically bind toTSLP. In one embodiment, the at least two ISVDs specifically binding toIL-13 specifically bind to human IL-13 and the at least two ISVDsspecifically binding to TSLP specifically bind to human TSLP. In oneembodiment, the at least two ISVDs specifically binding to IL-13 aredistinct ISVDs and the at least two ISVDs binding to TSLP are distinctISVDs. In another embodiment, the polypeptide comprising or consistingof at least four ISVDs further comprises one or more other groups,residues, moieties or binding units, optionally linked via one or morepeptidic linkers, in which said one or more other groups, residues,moieties or binding units provide the polypeptide with increasedhalf-life, compared to the corresponding polypeptide without said one ormore other groups, residues, moieties or binding units. For example, thebinding unit can be an ISVD that binds to a (human) serum protein, suchas human serum albumin.

Also provided is a nucleic acid molecule capable of expressing thepolypeptide of the present technology, a nucleic acid or vectorcomprising the nucleic acid, and a composition comprising thepolypeptide, the nucleic acid or the vector. In one embodiment, thecomposition is a pharmaceutical composition.

Also provided is a host or host cell comprising the nucleic acid orvector that encodes the polypeptide according to the present technology.

Further provided is a method for producing the polypeptide according topresent technology, said method at least comprising the steps of:

-   -   a. expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid comprising a        nucleotide sequence that encodes a polypeptide of the present        technology; optionally followed by:    -   b. isolating and/or purifying the polypeptide according to the        present technology.

Moreover, the present technology provides the polypeptide, thecomposition comprising the polypeptide, or the composition comprisingthe nucleic acid or vector comprising the nucleotide sequence thatencodes the polypeptide, for use as a medicament. In one embodiment, thepolypeptide or composition is for use in the treatment of aninflammatory disease, such as a type 2 inflammatory disease. In oneembodiment, the type 2 inflammatory disease is selected from atopicdermatitis and asthma.

In addition, provided is a method of treating an inflammatory disease,such as a type 2 inflammatory disease, wherein said method comprisesadministering, to a subject in need thereof, a pharmaceutically activeamount of the polypeptide or a composition according to the presenttechnology. In one embodiment, the type 2 inflammatory disease isselected from atopic dermatitis and asthma. In one embodiment, themethod further comprises administering one or more additionaltherapeutic agents.

Further provided is the use of the polypeptide or composition of thepresent technology in the preparation of a pharmaceutical compositionfor treating an inflammatory disease, such as a type 2 inflammatorydisease. In one embodiment, the type 2 inflammatory disease is selectedfrom atopic dermatitis and asthma.

In particular, the present technology provides the followingembodiments:

-   Embodiment 1. A polypeptide, a composition comprising the    polypeptide, or a composition comprising a nucleic acid comprising a    nucleotide sequence that encodes the polypeptide, for use as a    medicament, wherein the polypeptide comprises or consists of at    least one immunoglobulin single variable domain (ISVD), wherein said    ISVD comprises three complementarity determining regions (CDR1 to    CDR3, respectively), and wherein the at least one ISVD comprises:    -   a) a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 7;        -   a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 12; and        -   a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 17,    -   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 8;        -   a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 13; and        -   a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 18,    -   c) a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 9;        -   a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 14; and        -   a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 19, or    -   d) a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 11;        -   a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 16; and        -   a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 21.-   Embodiment 2. The polypeptide or composition for use according to    embodiment 1, wherein the at least one ISVD comprises:    -   a) a CDR1 that is the amino acid sequence of SEQ ID NO: 7, a        CDR2 that is the amino acid sequence of SEQ ID NO: 12 and a CDR3        that is the amino acid sequence of SEQ ID NO: 17,    -   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a        CDR2 that is the amino acid sequence of SEQ ID NO: 13 and a CDR3        that is the amino acid sequence of SEQ ID NO: 18,    -   c) a CDR1 that is the amino acid sequence of SEQ ID NO: 9, a        CDR2 that is the amino acid sequence of SEQ ID NO: 14 and a CDR3        that is the amino acid sequence of SEQ ID NO: 19, or    -   d) a CDR1 that is the amino acid sequence of SEQ ID NO: 11, a        CDR2 that is the amino acid sequence of SEQ ID NO: 16 and a CDR3        that is the amino acid sequence of SEQ ID NO: 21.-   Embodiment 3. The polypeptide or composition for use according to    any of embodiments 1 or 2, wherein the amino acid sequence of the at    least one ISVD comprises:    -   a) a sequence identity of more than 90% with SEQ ID NO: 2,    -   b) a sequence identity of more than 90% with SEQ ID NO: 3,    -   c) a sequence identity of more than 90% identity with SEQ ID NO:        4, or    -   d) a sequence identity of more than 90% identity with SEQ ID NO:        6.-   Embodiment 4. The polypeptide or composition for use according to    any of embodiments 1 to 3, wherein said at least one ISVD comprises:    -   a) the amino acid sequence of SEQ ID NO: 2,    -   b) the amino acid sequence of SEQ ID NO: 3,    -   c) the amino acid sequence of SEQ ID NO: 4, or    -   d) the amino acid sequence of SEQ ID NO: 6.-   Embodiment 5. The polypeptide or composition for use according to    embodiment 1, wherein the polypeptide comprises or consists of at    least two ISVDs, wherein each of said ISVDs comprises three    complementarity determining regions (CDR1 to CDR3, respectively),    wherein the at least two ISVDs are optionally linked via one or more    peptidic linkers, and wherein:    -   a) a first and a second ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 7;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 12; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 17,    -   b) a first and a second ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 8;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 13            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 13; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 18,    -   c) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 7;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 12; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 17, and    -    a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 8            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 8;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 13            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 13; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 18            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 18,    -   d) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 8;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 13; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 18, and    -    a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 7            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 7;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 12            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 12; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 17            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 17,    -   e) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 11            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 11;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 16            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 16; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 21, and    -    a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 9            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 9;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 14            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 14; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 19            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 19, or    -   f) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 9;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 14            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 14; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 19            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 19, and/or    -    a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 11            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 11;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 16            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 16; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 21            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 21,    -   wherein the order of the ISVDs indicates their relative position        to each other considered from the N-terminus to the C-terminus        of said polypeptide.-   Embodiment 6. The polypeptide or composition for use according to    embodiment 5, wherein:    -   a) the first and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino        acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 17,    -   b) the first and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino        acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 18,    -   c) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence        of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 17, and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino        acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 18,    -   d) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence        of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 18, and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino        acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 17,    -   e) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 11, a CDR2 that is the amino acid        sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21, and the second ISVD comprises a CDR1        that is the amino acid sequence of SEQ ID NO: 9, a CDR2 that is        the amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 19, or    -   f) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence        of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 19, and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 11, a CDR2 that is the amino        acid sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21.-   Embodiment 7. The polypeptide or composition for use according to    any of embodiments 5 or 6, wherein:    -   a) the amino acid sequence of the first and the second ISVD        comprises a sequence identity of more than 90% with SEQ ID NO:        2,    -   b) the amino acid sequence of the first and the second ISVD        comprises a sequence identity of more than 90% with SEQ ID NO:        3,    -   c) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 2, and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 3,    -   d) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 3, and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 2,    -   e) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 6, and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 4,    -   f) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 4, and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 6.-   Embodiment 8. The polypeptide or composition for use according to    any of embodiments 5 to 7, wherein:    -   a) the first and the second ISVD comprises the amino acid        sequence of SEQ ID NO: 2,    -   b) the first and the second ISVD comprises the amino acid        sequence of SEQ ID NO: 3,    -   c) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 2, and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 3,    -   d) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 3, and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 2,    -   e) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 6, and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 4, or    -   f) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 4, and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 6.-   Embodiment 9. The polypeptide according to any of embodiments 5 to    8, wherein the polypeptide comprises or consists of:    -   a) the amino acid sequence of SEQ ID NO: 148,    -   b) the amino acid sequence of SEQ ID NO: 149,    -   c) the amino acid sequence of SEQ ID NO: 150,    -   d) the amino acid sequence of SEQ ID NO: 151,    -   e) the amino acid sequence of SEQ ID NO: 152,    -   f) the amino acid sequence of SEQ ID NO: 153,    -   g) the amino acid sequence of SEQ ID NO: 154,    -   h) the amino acid sequence of SEQ ID NO: 155,    -   i) the amino acid sequence of SEQ ID NO: 156,    -   j) the amino acid sequence of SEQ ID NO: 157,    -   k) the amino acid sequence of SEQ ID NO: 158, or    -   l) the amino acid sequence of SEQ ID NO: 159.-   Embodiment 10. The polypeptide or composition for use according to    any of embodiments 1 or 5, wherein the polypeptide comprises or    consists of at least four ISVDs, wherein each of said ISVDs    comprises three complementarity determining regions (CDR1 to CDR3,    respectively), wherein the at least four ISVDs are optionally linked    via one or more peptidic linkers, and wherein:    -   a) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 7;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 12; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 17;    -   b) a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 8            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 8;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 13            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 13; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 18            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 18;    -   c) a third ISVD comprises        -   vii. a CDR1 that is the amino acid sequence of SEQ ID NO: 9            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 9;        -   viii. a CDR2 that is the amino acid sequence of SEQ ID NO:            14 or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 14; and        -   ix. a CDR3 that is the amino acid sequence of SEQ ID NO: 19            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 19; and    -   d) a fourth ISVD comprises        -   x. a CDR1 that is the amino acid sequence of SEQ ID NO: 11            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 11;        -   xi. a CDR2 that is the amino acid sequence of SEQ ID NO: 16            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 16; and        -   xii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 21, wherein the order of the            ISVDs indicates their relative position to each other            considered from the N-terminus to the C-terminus of said            polypeptide.-   Embodiment 11. The composition for use according to any one of    embodiments 1 to 10, which is a pharmaceutical composition which    further comprises at least one pharmaceutically acceptable carrier,    diluent or excipient and/or adjuvant, and optionally comprises one    or more further pharmaceutically active polypeptides and/or    compounds.-   Embodiment 12. The polypeptide or composition for use according to    embodiment 10 or 11, wherein:    -   a) said first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence        of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 17;    -   b) said second ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence        of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 18;    -   c) said third ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence        of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 19; and    -   d) said fourth ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 11, a CDR2 that is the amino acid        sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21.-   Embodiment 13. The polypeptide or composition for use according to    any of embodiments 10 to 12, wherein:    -   a) the amino acid sequence of said first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 2;    -   b) the amino acid sequence of said second ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 3;    -   c) the amino acid sequence of said third ISVD comprises a        sequence identity of more than 90% identity with SEQ ID NO: 4;        and    -   d) the amino acid sequence of said fourth ISVD comprises a        sequence identity of more than 90% identity with SEQ ID NO: 6.-   Embodiment 14. The polypeptide or composition for use according to    any of embodiments 10 to 13, wherein:    -   a) said first ISVD comprises the amino acid sequence of SEQ ID        NO: 2;    -   b) said second ISVD comprises the amino acid sequence of SEQ ID        NO: 3;    -   c) said third ISVD comprises the amino acid sequence of SEQ ID        NO: 4; and    -   d) said fourth ISVD comprises the amino acid sequence of SEQ ID        NO: 6.-   Embodiment 15. The polypeptide or composition for use according to    any of embodiments 1 to 14, wherein said polypeptide further    comprises one or more other groups, residues, moieties or binding    units, optionally linked via one or more peptidic linkers, in which    said one or more other groups, residues, moieties or binding units    provide the polypeptide with increased half-life, compared to the    corresponding polypeptide without said one or more other groups,    residues, moieties or binding units.-   Embodiment 16. The polypeptide or composition for use according to    embodiment 15, in which said one or more other groups, residues,    moieties or binding units that provide the polypeptide with    increased half-life is chosen from the group consisting of a    polyethylene glycol molecule, serum proteins or fragments thereof,    binding units that can bind to serum proteins, an Fc portion, and    small proteins or peptides that can bind to serum proteins.-   Embodiment 17. The polypeptide or composition for use according to    any one of embodiments 15 to 16, in which said one or more other    binding units that provide the polypeptide with increased half-life    is chosen from the group consisting of binding units that can bind    to serum albumin (such as human serum albumin) or a serum    immunoglobulin (such as IgG).-   Embodiment 18. The polypeptide or composition for use according to    embodiment 17, in which said binding unit that provides the    polypeptide with increased half-life is an ISVD that can bind to    human serum albumin.-   Embodiment 19. The polypeptide or composition for use according to    embodiment 18, wherein the ISVD binding to human serum albumin    comprises    -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 10 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 10;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 15 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 15; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 20 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 20.-   Embodiment 20. The polypeptide or composition for use according to    any of embodiments 18 to 19, wherein the ISVD binding to human serum    albumin comprises a CDR1 that is the amino acid sequence of SEQ ID    NO: 10, a CDR2 that is the amino acid sequence of SEQ ID NO: 15 and    a CDR3 that is the amino acid sequence of SEQ ID NO: 20.-   Embodiment 21. The polypeptide or composition for use according to    any of embodiments 18 to 20, wherein the amino acid sequence of said    ISVD binding to human serum albumin comprises a sequence identity of    more than 90% with SEQ ID NO: 5.-   Embodiment 22. The polypeptide or composition for use according to    any of embodiments 18 to 21, wherein said ISVD binding to human    serum albumin comprises the amino acid sequence of SEQ ID NO: 5.-   Embodiment 23. The polypeptide or composition for use according to    any of embodiments 10 to 22, wherein the amino acid sequence of the    polypeptide comprises a sequence identity of more than 90% with SEQ    ID NO: 1.-   Embodiment 24. The polypeptide or composition for use according to    any of embodiments 10 to 23, wherein the polypeptide comprises or    consists of the amino acid sequence of SEQ ID NO: 1.-   Embodiment 25. The polypeptide or composition for use according to    any of embodiments 1 to 24, for use in the treatment of an    inflammatory disease, such as a type 2 inflammatory disease.-   Embodiment 26. The polypeptide or composition for use according to    embodiment 25, wherein the type 2 inflammatory disease is selected    from asthma and atopic dermatitis.-   Embodiment 27. A polypeptide that comprises or consists of at least    one immunoglobulin single variable domain (ISVD), wherein said ISVD    comprises three complementarity determining regions (CDR1 to CDR3,    respectively); and wherein the at least one ISVD comprises:    -   a) a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 7;        -   a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 12; and        -   a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 17,    -   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 8;        -   a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 13; and        -   a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 18,    -   c) a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 9;        -   a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 14; and        -   a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 19, or    -   d) a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 11;        -   a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 16; and        -   a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 21.-   Embodiment 28. The polypeptide according to embodiment 27, wherein    the at least one ISVD comprises:    -   a) a CDR1 that is the amino acid sequence of SEQ ID NO: 7, a        CDR2 that is the amino acid sequence of SEQ ID NO: 12 and a CDR3        that is the amino acid sequence of SEQ ID NO: 17,    -   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a        CDR2 that is the amino acid sequence of SEQ ID NO: 13 and a CDR3        that is the amino acid sequence of SEQ ID NO: 18,    -   c) a CDR1 that is the amino acid sequence of SEQ ID NO: 9, a        CDR2 that is the amino acid sequence of SEQ ID NO: 14 and a CDR3        that is the amino acid sequence of SEQ ID NO: 19, or    -   d) a CDR1 that is the amino acid sequence of SEQ ID NO: 11, a        CDR2 that is the amino acid sequence of SEQ ID NO: 16 and a CDR3        that is the amino acid sequence of SEQ ID NO: 21.-   Embodiment 29. The polypeptide according to any of embodiments 27 or    28, wherein the amino acid sequence of the at least one ISVD    comprises:    -   a) a sequence identity of more than 90% with SEQ ID NO: 2,    -   b) a sequence identity of more than 90% with SEQ ID NO: 3,    -   c) a sequence identity of more than 90% identity with SEQ ID NO:        4, or    -   d) a sequence identity of more than 90% identity with SEQ ID NO:        6.-   Embodiment 30. The polypeptide according to any of embodiments 27 to    29, wherein said at least one ISVD comprises:    -   a) the amino acid sequence of SEQ ID NO: 2,    -   b) the amino acid sequence of SEQ ID NO: 3,    -   c) the amino acid sequence of SEQ ID NO: 4, or    -   d) the amino acid sequence of SEQ ID NO: 6.-   Embodiment 31. The polypeptide according to embodiment 1 or 27,    wherein the polypeptide comprises or consists of at least two ISVDs,    wherein each of said ISVDs comprises three complementarity    determining regions (CDR1 to CDR3, respectively), wherein the at    least two ISVDs are optionally linked via one or more peptidic    linkers, and wherein:    -   a) a first and a second ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 7;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 12; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 17,    -   b) a first and a second ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 8;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 13            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 13; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 18,    -   c) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 7;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 12; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 17, and    -    a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 8            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 8;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 13            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 13; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 18            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 18,    -   d) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 8;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 13            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 13; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 18, and    -    a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 7            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 7;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 12            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 12; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 17            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 17,    -   e) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 11            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 11;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 16            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 16; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 21, and    -    a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 9            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 9;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 14            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 14; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 19            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 19, or    -   f) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 9;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 14            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 14; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 19            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 19, and    -    a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 11            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 11;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 16            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 16; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 21            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 21,    -   wherein the order of the ISVDs indicates their relative position        to each other considered from the N-terminus to the C-terminus        of said polypeptide.-   Embodiment 32. The polypeptide according to embodiment 31, wherein:    -   a) the first and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino        acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 17,    -   b) the first and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino        acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 18,    -   c) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence        of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 17, and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino        acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 18,    -   d) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence        of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 18, and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino        acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 17,    -   e) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 11, a CDR2 that is the amino acid        sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21, and the second ISVD comprises a CDR1        that is the amino acid sequence of SEQ ID NO: 9, a CDR2 that is        the amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 19, or    -   f) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence        of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 19, and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 11, a CDR2 that is the amino        acid sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21.-   Embodiment 33. The polypeptide according to any of embodiments 31 or    32, wherein:    -   a) the amino acid sequence of the first and the second ISVD        comprises a sequence identity of more than 90% with SEQ ID NO:        2,    -   b) the amino acid sequence of the first and the second ISVD        comprises a sequence identity of more than 90% with SEQ ID NO:        3,    -   c) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 2, and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 3,    -   d) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 3, and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 2,    -   e) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 6, and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 4,    -   f) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 4, and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 6.-   Embodiment 34. The polypeptide according to any of embodiments 31 to    33, wherein:    -   a) the first and the second ISVD comprises the amino acid        sequence of SEQ ID NO: 2,    -   b) the first and the second ISVD comprises the amino acid        sequence of SEQ ID NO: 3,    -   c) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 2, and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 3,    -   d) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 3, and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 2,    -   e) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 6, and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 4, or    -   f) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 4, and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 6.-   Embodiment 35. The polypeptide according to any of embodiments 31 to    34, wherein the polypeptide comprises or consists of:    -   a) the amino acid sequence of SEQ ID NO: 148,    -   b) the amino acid sequence of SEQ ID NO: 149,    -   c) the amino acid sequence of SEQ ID NO: 150,    -   d) the amino acid sequence of SEQ ID NO: 151,    -   e) the amino acid sequence of SEQ ID NO: 152,    -   f) the amino acid sequence of SEQ ID NO: 153,    -   g) the amino acid sequence of SEQ ID NO: 154,    -   h) the amino acid sequence of SEQ ID NO: 155,    -   i) the amino acid sequence of SEQ ID NO: 156,    -   j) the amino acid sequence of SEQ ID NO: 157,    -   k) the amino acid sequence of SEQ ID NO: 158, or    -   l) the amino acid sequence of SEQ ID NO: 159.-   Embodiment 36. The polypeptide according to any of embodiments 27 or    31, wherein the polypeptide comprises or consists of at least four    ISVDs, wherein each of said ISVDs comprises three complementarity    determining regions (CDR1 to CDR3, respectively), wherein the at    least four ISVDs are optionally linked via one or more peptidic    linkers, and wherein:    -   a) a first ISVD comprises        -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or            an amino acid sequence with 2 or 1 amino acid difference(s)            with SEQ ID NO: 7;        -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 12; and        -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 17;    -   b) a second ISVD comprises        -   iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 8            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 8;        -   v. a CDR2 that is the amino acid sequence of SEQ ID NO: 13            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 13; and        -   vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 18            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 18;    -   c) a third ISVD comprises        -   vii. a CDR1 that is the amino acid sequence of SEQ ID NO: 9            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 9;        -   viii. a CDR2 that is the amino acid sequence of SEQ ID NO:            14 or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 14; and        -   ix. a CDR3 that is the amino acid sequence of SEQ ID NO: 19            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 19; and    -   d) a fourth ISVD comprises        -   x. a CDR1 that is the amino acid sequence of SEQ ID NO: 11            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 11;        -   xi. a CDR2 that is the amino acid sequence of SEQ ID NO: 16            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 16; and        -   xii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21            or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 21,    -   wherein the order of the ISVDs indicates their relative position        to each other considered from the N-terminus to the C-terminus        of said polypeptide.-   Embodiment 37. The polypeptide according to embodiment 36, wherein:    -   a) said first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence        of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 17;    -   b) said second ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence        of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 18;    -   c) said third ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence        of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 19; and    -   d) said fourth ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 11, a CDR2 that is the amino acid        sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21.-   Embodiment 38. The polypeptide according to any of embodiments 36 or    37, wherein:    -   a) the amino acid sequence of said first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 2;    -   b) the amino acid sequence of said second ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 3;    -   c) the amino acid sequence of said third ISVD comprises a        sequence identity of more than 90% identity with SEQ ID NO: 4;        and    -   d) the amino acid sequence of said fourth ISVD comprises a        sequence identity of more than 90% identity with SEQ ID NO: 6.-   Embodiment 39. The polypeptide according to any of embodiments 36 to    38, wherein:    -   a) said first ISVD comprises the amino acid sequence of SEQ ID        NO: 2;    -   b) said second ISVD comprises the amino acid sequence of SEQ ID        NO: 3;    -   c) said third ISVD comprises the amino acid sequence of SEQ ID        NO: 4; and    -   d) said fourth ISVD comprises the amino acid sequence of SEQ ID        NO: 6.-   Embodiment 40. The polypeptide according to any of embodiments 27 to    39, wherein said polypeptide further comprises one or more other    groups, residues, moieties or binding units, optionally linked via    one or more peptidic linkers, in which said one or more other    groups, residues, moieties or binding units provide the polypeptide    with increased half-life, compared to the corresponding polypeptide    without said one or more other groups, residues, moieties or binding    units.-   Embodiment 41. The polypeptide according to embodiment 40, in which    said one or more other groups, residues, moieties or binding units    that provide the polypeptide with increased half-life is chosen from    the group consisting of a polyethylene glycol molecule, serum    proteins or fragments thereof, binding units that can bind to serum    proteins, an Fc portion, and small proteins or peptides that can    bind to serum proteins.-   Embodiment 42. The polypeptide according to any one of embodiments    40 to 41, in which said one or more other binding units that provide    the polypeptide with increased half-life is chosen from the group    consisting of binding units that can bind to serum albumin (such as    human serum albumin) or a serum immunoglobulin (such as IgG).-   Embodiment 43. The polypeptide according to embodiment 42, in which    said binding unit that provides the polypeptide with increased    half-life is an ISVD that can bind to human serum albumin.-   Embodiment 44. The polypeptide according to embodiment 43, wherein    the ISVD binding to human serum albumin comprises    -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 10 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 10;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 15 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 15; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 20 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 20.-   Embodiment 45. The polypeptide according to any of embodiments 43 to    44, wherein the ISVD binding to human serum albumin comprises a CDR1    that is the amino acid sequence of SEQ ID NO:10, a CDR2 that is the    amino acid sequence of SEQ ID NO: 15 and a CDR3 that is the amino    acid sequence of SEQ ID NO: 20.-   Embodiment 46. The polypeptide according to any of embodiments 43 to    45, wherein the amino acid sequence of said ISVD binding to human    serum albumin comprises a sequence identity of more than 90% with    SEQ ID NO: 5.-   Embodiment 47. The polypeptide according to any of embodiments 43 to    46, wherein said ISVD binding to human serum albumin comprises the    amino acid sequence of SEQ ID NO: 5.-   Embodiment 48. The polypeptide according to any of embodiments 36 to    47, wherein the amino acid sequence of the polypeptide comprises a    sequence identity of more than 90% with SEQ ID NO: 1.-   Embodiment 49. The polypeptide according to any of embodiments 36 to    48, wherein the polypeptide comprises or consists of the amino acid    sequence of SEQ ID NO: 1.-   Embodiment 50. A nucleic acid comprising a nucleotide sequence that    encodes a polypeptide according to any of embodiments 27 to 49, or a    polypeptide according to embodiments 36 to 49.-   Embodiment 51. A host or host cell comprising a nucleic acid    according to embodiment 50.-   Embodiment 52. A method for producing a polypeptide according to any    of embodiments 27 to 49, or a polypeptide according to embodiments    36 to 49, said method at least comprising the steps of:    -   a) expressing, in a suitable host cell or host organism or in        another suitable expression system, a nucleic acid according to        embodiment 50; optionally followed by:    -   b) isolating and/or purifying the polypeptide according to any        of embodiments 27 to 49, or the polypeptide according to        embodiments 36 to 49.-   Embodiment 53. A composition comprising at least one polypeptide    according to any of embodiments 27 to 49, or at least one    polypeptide according to embodiments 36 to 49, or a nucleic acid    according to embodiment 50.-   Embodiment 54. The composition according to embodiment 53, which is    a pharmaceutical composition which further comprises at least one    pharmaceutically acceptable carrier, diluent or excipient and/or    adjuvant, and optionally comprises one or more further    pharmaceutically active polypeptides and/or compounds.-   Embodiment 55. A method of treating an inflammatory disease, such as    a type 2 inflammatory disease, wherein said method comprises    administering, to a subject in need thereof, a pharmaceutically    active amount of a polypeptide according to any of embodiments 27 to    49, or according to embodiments 36 to 49, or a composition according    to any of embodiments 53 to 54.-   Embodiment 56. The method according to embodiment 55, wherein the    type 2 inflammatory disease is selected from asthma and atopic    dermatitis.-   Embodiment 57. Use of a polypeptide according to any of embodiments    27 to 49, or according to embodiments 36 to 49, or a composition    according to any of embodiments 53 to 54, in the preparation of a    pharmaceutical composition for treating an inflammatory disease,    such as a type 2 inflammatory disease.-   Embodiment 58. Use of the polypeptide or a composition according to    embodiments 57, wherein the type 2 inflammatory disease is selected    from asthma and atopic dermatitis.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Sensorgram showing simultaneous binding of IL13 and HSA toF027400161 captured via TSLP.

FIG. 2: Inhibition of human, cyno and rhesus IL13 in the eotaxin releaseassay by V_(HH) F027400161 and the reference compounds anti-hIL-13 mAb1and anti-hIL-13 mAb2.

FIG. 3: Inhibition of human and cyno IL13 in the SEAP reporter assay byF027400161 and the reference compounds anti-hIL-13 mAb1 and anti-hIL-13mAb2.

FIG. 4: Inhibition of human and cyno TSLP induced BaF3 proliferation byF027400161 and the reference compound anti-hTSLP mAb1. IRR00096 is anegative control Nb.

FIG. 5: Box plot showing the binding of pre-existing antibodies presentin 96 human serum samples to F027400161, 163 and 164 compared to controlF027301186.

FIG. 6: Dose-response inhibition profiles of F-27400161 (also referredto as F027400161) and comparator antibody anti-hTSLP reference mAb1 onTSLP-induced CCL17 response in human DCs. Enriched human DCs weretreated with 4 ng/mL of TSLP and incubated with 8 concentrations of theISVD construct and anti-hTSLP reference mAb1 for 36 hours. CCL17concentration in freshly collected supernatant was measured by ELISA.IC₅₀ values were calculated by nonlinear regression (log inhibitor vsresponses−variable slope four parameters lease squares fit) in GraphpadPrism 8.0 with no constraints. Data are represented as mean±standarderror of mean (SEM) of all 8 donors combined from 8 individualexperiments.

FIG. 7: Dose inhibition responses of F-27400161 (also referred to asF027400161) and comparators Anti-hIL-13 reference mAb1 and anti-hTSLPreference mAb1 on 0.5 ng/mL IL-13 and TSLP-induced synergisticproduction of CCL17 in human PBMCs. Healthy donor PBMCs were stimulatedwith 0.5 ng/mL of recombinant IL-13+TSLP and incubated with 10 doses ofthe ISVD construct (Nb) and comparators for 20 hours. CCL17concentration in the cell culture supernatant was measured by MSD V-Plexkit. IC₅₀ values were calculated by nonlinear regression (log inhibitorvs responses−variable slope four parameters lease squares fit) inGraphpad Prism 8.0 with no constraints. Data are represented asmean±standard error of mean (SEM) of all donors combined from 8individual experiments.

FIG. 8: Dose inhibition responses of F-27400161 (also referred to asF027400161) and comparator antibodies anti-hIL-13 reference mAb1 andanti-hTSLP reference mAb1 on 5 ng/mL IL-13 and TSLP-induced synergisticproduction of CCL17 in human PBMCs. Healthy donor PBMCs were stimulatedwith 5 ng/mL of recombinant IL-13+TSLP and incubated with 10 doses ofthe ISVD construct (Nb) and comparator antibodies for 20 hours. CCL17concentration in freshly collected supernatant was measured by MSDV-Plex kit. IC₅₀ values were calculated by nonlinear regression (loginhibitor vs responses—variable slope four parameters lease squares fit)in Graphpad Prism 8.0 with no constraints. Data are represented asmean±standard error of mean (SEM) of all donors combined from 8individual experiments.

FIG. 9: Inhibition profiles of F-27400161 (also referred to asF027400161) and comparator antibodies anti-hIL-13 reference mAb1 andant-hTSLP reference mAb1 on allergen Der P-induced IL-5, CCL17, andCCL26 production by human PBMCs in a triculture assay. Normal donorPBMCs cocultured with MRCS fibroblasts and A549 epithelial cells werestimulated with 3 mg/mL of Der P, and incubated with 11.1 nM of theISVD, anti-hIL-13 reference mAb1, or anti-hTSLP reference mAb1 in a24-well plate for 6 days in a 37° C. cell-culture incubator. IL-5,CCL17, and CCL26 concentration in freshly collected supernatant wasmeasured by Human Magnetic Luminex Assays. Percentage of inhibition werecalculated relative to unstimulated (min) and stimulated (max) controlsamples which did not receive either ISVD polypeptides or antibodies.All calculations were performed using GraphPad Prism 8.0. Data arerepresented as mean±standard error of mean (SEM) of all donors combinedfrom 3 independent experiments.

FIG. 10: F027400161 significantly reduced detectable levels of humanTSLP in the plasma of NSG-SGM3 mice. When compared to vehicle treatedmice (376.166 pg/ml), human plasma TSLP levels were reduced with the 0.1mg/kg F027400161 dose (45.772 pg/ml) and the 10 mg/kg F027400161 dose(0.072 pg/ml), demonstrating that the TSLP arm of F027400161 can bind tohuman TSLP in the plasma of humanized NSG-SGM3 mice.

FIG. 11: F027400161 significantly reduced detectable levels of humanIL-13 in the plasma of NSG-SGM3 mice. When compared to vehicle treatedmice (274.052 pg/ml), human IL-13 levels were reduced with the 0.01mg/kg F027400161 dose (201.286 pg/ml), 0.05 mg/kg F027400161 dose(22.028 pg/ml), 0.1 mg/kg F027400161 dose (40.740 pg/ml), and with the10 mg/kg F027400161 dose (1.777 pg/ml), demonstrating that the IL-13 armof F27400161 can bind to human IL-13 in the plasma of humanized NSG-SGM3mice.

FIGS. 12 and 13: F027400161 Significantly reduced mouse Retnla and Clca1transcript expression in the lungs of NSG-SGM3 mice that receivedhydrodynamic delivery of hTSLP and hIL-4. Overexpression of human TSLPand IL-4 in the NSG-SGM3 mice induces the production of hIL-13 fromhuman immune cells derived from the precursor CD34⁺. Neutralization ofthe human IL-13 by F027400161 resulted in the inhibition of the mouseRetnla and Clca1 marker genes at the 10 mg/Kg dose.

FIG. 14: Schematic presentation of ISVD construct F027400161 showingfrom the N-terminus to the C-terminus the monovalent buildingblocks/ISVDs 4B02, 4B06, 501A02, 529F10, as well as the albumin binderALB23002. Whereas 4B02 and 4B06 are connected via a 35GS linker, theremaining building block are linked via 9GS linkers.

5. DETAILED DESCRIPTION OF THE PRESENT TECHNOLOGY

The present technology aims at providing a novel type of drug fortreating inflammatory diseases, such as atopic dermatitis and asthma.

In some embodiments, the present technology relates to a polypeptidetargeting IL-13 and TSLP at the same time leading to an increasedefficiency of modulating a type 2 inflammatory response as compared tomonospecific anti-IL-13 or anti-TSLP polypeptides in vitro and/or invivo. In some embodiments, the polypeptides are efficiently produced(e.g. in microbial hosts). Furthermore, in some embodiments, suchpolypeptides could be shown to have limited reactivity to pre-existingantibodies in the subject to be treated (i.e. antibodies present in thesubject before the first treatment with the antibody construct). Inother embodiments such polypeptides exhibit a half-life in the subjectto be treated that is long enough such that consecutive treatments canbe conveniently spaced apart.

5.1 The Polypeptides of the Present Technology

Monospecific-Monovalent Polypeptides

In one embodiment, the polypeptide of the present technology ismonospecific and monovalent.

The term “monospecific” refers to the binding to one (specific) type oftarget molecule(s). A monospecific polypeptide of the present technologythus specifically binds to IL-13. Another monospecific polypeptide ofthe present technology specifically binds to TSLP.

The term “monovalent” indicates the presence of only one bindingunits/building block that (specifically) targets a molecule, such asISVDs.

Accordingly, in one aspect, the present technology provides amonospecific-monovalent polypeptide comprising or consisting of one ISVDthat specifically binds to IL-13, which comprises three complementaritydetermining regions (CDR1 to CDR3, respectively). The ISVD can beselected from an ISVD comprising:

-   -   a) a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 7, a CDR2 that is the amino acid sequence of SEQ ID        NO: 12 or an amino acid sequence with 2 or 1 amino acid        difference(s) with SEQ ID NO: 12, and a CDR3 that is the amino        acid sequence of SEQ ID NO: 17 or an amino acid sequence with 2        or 1 amino acid difference(s) with SEQ ID NO: 17; or    -   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID        NO: 13 or an amino acid sequence with 2 or 1 amino acid        difference(s) with SEQ ID NO: 13, and a CDR3 that is the amino        acid sequence of SEQ ID NO: 18 or an amino acid sequence with 2        or 1 amino acid difference(s) with SEQ ID NO: 18.

In one embodiment of this aspect of the present technology, the ISVDspecifically binds to human IL-13.

In a further embodiment, the ISVD specifically binding to IL-13 isselected from an ISVD comprising:

-   -   a) a CDR1 that is the amino acid sequence of SEQ ID NO: 7, a        CDR2 that is the amino acid sequence of SEQ ID NO: 12 and a CDR3        that is the amino acid sequence of SEQ ID NO: 17; or    -   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a        CDR2 that is the amino acid sequence of SEQ ID NO: 13 and a CDR3        that is the amino acid sequence of SEQ ID NO: 18.

In a further embodiment of this aspect of the present technology, theISVD specifically binding to IL-13 is selected from an ISVD comprising:

-   -   a) a sequence identity of more than 90% with SEQ ID NO: 2; or    -   b) a sequence identity of more than 90% with SEQ ID NO: 3.

In one embodiment, the ISVD specifically binding to IL-13 is selectedfrom an ISVD comprising the amino acid sequence of SEQ ID NO: 2; or theamino acid sequence of SEQ ID NO: 3.

In another aspect the present technology provides amonospecific-monovalent polypeptide comprising or consisting of one ISVDthat specifically binds to TSLP, which comprises three complementaritydetermining regions (CDR1 to CDR3, respectively). The ISVD can beselected from an ISVD comprising:

-   -   a) a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 9, a CDR2 that is the amino acid sequence of SEQ ID        NO: 14 or an amino acid sequence with 2 or 1 amino acid        difference(s) with SEQ ID NO: 14, and a CDR3 that is the amino        acid sequence of SEQ ID NO: 19 or an amino acid sequence with 2        or 1 amino acid difference(s) with SEQ ID NO: 19; or    -   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 11, a CDR2 that is the amino acid sequence of SEQ ID        NO: 16 or an amino acid sequence with 2 or 1 amino acid        difference(s) with SEQ ID NO: 16, and a CDR3 that is the amino        acid sequence of SEQ ID NO: 21 or an amino acid sequence with 2        or 1 amino acid difference(s) with SEQ ID NO: 21.

In one embodiment of this aspect of the present technology, the ISVDspecifically binds to human TSLP.

In a further embodiment, the ISVD specifically binding to TSLP isselected from an ISVD comprising:

-   -   a) a CDR1 that is the amino acid sequence of SEQ ID NO: 9, a        CDR2 that is the amino acid sequence of SEQ ID NO: 14 and a CDR3        that is the amino acid sequence of SEQ ID NO: 19; or    -   b) a CDR1 that is the amino acid sequence of SEQ ID NO: 11, a        CDR2 that is the amino acid sequence of SEQ ID NO: 16 and a CDR3        that is the amino acid sequence of SEQ ID NO: 21.

In a further embodiment of this aspect of the present technology, theISVD specifically binding to TSLP is selected from an ISVD comprising:

-   -   a) a sequence identity of more than 90% with SEQ ID NO: 4; or    -   b) a sequence identity of more than 90% with SEQ ID NO: 6.

In one embodiment, the ISVD specifically binding to TSLP is selectedfrom an ISVD comprising the amino acid sequence of SEQ ID NO: 4; or theamino acid sequence of SEQ ID NO: 6.

Monospecific-Multivalent Polypeptides

In another aspect, the polypeptide of the present technology ismonospecific and at least bivalent, but can also be e.g., trivalent,tetravalent, pentavalent, hexavalent, etc.

The terms “bivalent”, “trivalent”, “tetravalent”, “pentavalent”, or“hexavalent” all fall under the term “multivalent” and indicate thepresence of two, three, four, five or six binding units/building blocks,respectively, such as ISVDs.

Accordingly, in one aspect the present technology provides amonospecific-bivalent polypeptide comprising or consisting of two ISVDsthat specifically bind to IL-13, wherein each of the two ISVDs comprisesthree complementarity determining regions (CDR1 to CDR3, respectively),wherein the two ISVDs are optionally linked via one or more peptidiclinkers, and wherein:

-   -   a) a first and a second ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 7 or an amino acid sequence with 2        or 1 amino acid difference(s) with SEQ ID NO: 7, a CDR2 that is        the amino acid sequence of SEQ ID NO: 12 or an amino acid        sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:        12, and a CDR3 that is the amino acid sequence of SEQ ID NO: 17        or an amino acid sequence with 2 or 1 amino acid difference(s)        with SEQ ID NO: 17;    -   b) a first and a second ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 8 or an amino acid sequence with 2        or 1 amino acid difference(s) with SEQ ID NO: 8, a CDR2 that is        the amino acid sequence of SEQ ID NO: 13 or an amino acid        sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:        13, and a CDR3 that is the amino acid sequence of SEQ ID NO: 18        or an amino acid sequence with 2 or 1 amino acid difference(s)        with SEQ ID NO: 18;    -   c) a first ISVD comprises a CDR1 that is the amino acid sequence        of SEQ ID NO: 7 or an amino acid sequence with 2 or 1 amino acid        difference(s) with SEQ ID NO: 7, a CDR2 that is the amino acid        sequence of SEQ ID NO: 12 or an amino acid sequence with 2 or 1        amino acid difference(s) with SEQ ID NO: 12, and a CDR3 that is        the amino acid sequence of SEQ ID NO: 17 or an amino acid        sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:        17, and        -   a second ISVD comprises a CDR1 that is the amino acid            sequence of SEQ ID NO: 8 or an amino acid sequence with 2 or            1 amino acid difference(s) with SEQ ID NO: 8, a CDR2 that is            the amino acid sequence of SEQ ID NO: 13 or an amino acid            sequence with 2 or 1 amino acid difference(s) with SEQ ID            NO: 13, and a CDR3 that is the amino acid sequence of SEQ ID            NO: 18 or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 18; or    -   d) a first ISVD comprises a CDR1 that is the amino acid sequence        of SEQ ID NO: 8 or an amino acid sequence with 2 or 1 amino acid        difference(s) with SEQ ID NO: 8, a CDR2 that is the amino acid        sequence of SEQ ID NO: 13 or an amino acid sequence with 2 or 1        amino acid difference(s) with SEQ ID NO: 13, and a CDR3 that is        the amino acid sequence of SEQ ID NO: 18 or an amino acid        sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:        18, and        -   a second ISVD comprises a CDR1 that is the amino acid            sequence of SEQ ID NO: 7 or an amino acid sequence with 2 or            1 amino acid difference(s) with SEQ ID NO: 7, a CDR2 that is            the amino acid sequence of SEQ ID NO: 12 or an amino acid            sequence with 2 or 1 amino acid difference(s) with SEQ ID            NO: 12, and a CDR3 that is the amino acid sequence of SEQ ID            NO: 17 or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 17.

In one embodiment of this aspect of the present technology, the ISVDsare linked via one or more peptidic linkers. In one embodiment, the twoISVDs specifically bind human IL13.

In a further embodiment, the monospecific-bivalent polypeptide comprisesor consists of two ISVDs that specifically bind to IL-13, wherein:

-   -   a) the first and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino        acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 17;    -   b) the first and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino        acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 18;    -   c) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence        of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 17, and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino        acid sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 18; or    -   d) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence        of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 18, and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO: 7, a CDR2 that is the amino        acid sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 17.

In a further embodiment of this aspect of the present technology, themonospecific-bivalent polypeptide comprises or consists of two ISVDsthat specifically bind to IL-13, wherein:

-   -   a) the amino acid sequence of the first and the second ISVD        comprises a sequence identity of more than 90% with SEQ ID NO:        2;    -   b) the amino acid sequence of the first and the second ISVD        comprises a sequence identity of more than 90% with SEQ ID NO:        3;    -   c) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 2 and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 3; or    -   d) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 3 and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 2.

In one embodiment, the monospecific-bivalent polypeptide comprises orconsists of two ISVDs that specifically bind to IL-13, wherein:

-   -   a) the first and the second ISVD comprise the amino acid        sequence of SEQ ID NO: 2;    -   b) the first and the second ISVD comprise the amino acid        sequence of SEQ ID NO: 3:    -   c) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 2 and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 3; or    -   d) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 3 and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 2.

In another aspect the present technology provides amonospecific-bivalent polypeptide comprising or consisting of two ISVDsthat specifically bind to TSLP, wherein each of the two ISVDs comprisesthree complementarity determining regions (CDR1 to CDR3, respectively),wherein the two ISVDs are optionally linked via one or more peptidiclinkers, and wherein:

-   -   a) a first ISVD comprises a CDR1 that is the amino acid sequence        of SEQ ID NO: 11 or an amino acid sequence with 2 or 1 amino        acid difference(s) with SEQ ID NO: 11, a CDR2 that is the amino        acid sequence of SEQ ID NO: 16 or an amino acid sequence with 2        or 1 amino acid difference(s) with SEQ ID NO: 16, and a CDR3        that is the amino acid sequence of SEQ ID NO: 21 or an amino        acid sequence with 2 or 1 amino acid difference(s) with SEQ ID        NO: 21, and        -   a second ISVD comprises a CDR1 that is the amino acid            sequence of SEQ ID NO: 9 or an amino acid sequence with 2 or            1 amino acid difference(s) with SEQ ID NO: 9, a CDR2 that is            the amino acid sequence of SEQ ID NO: 14 or an amino acid            sequence with 2 or 1 amino acid difference(s) with SEQ ID            NO: 14, and a CDR3 that is the amino acid sequence of SEQ ID            NO: 19 or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 19, or    -   b) a first ISVD comprises a CDR1 that is the amino acid sequence        of SEQ ID NO: 9 or an amino acid sequence with 2 or 1 amino acid        difference(s) with SEQ ID NO: 9, a CDR2 that is the amino acid        sequence of SEQ ID NO: 14 or an amino acid sequence with 2 or 1        amino acid difference(s) with SEQ ID NO: 14, and a CDR3 that is        the amino acid sequence of SEQ ID NO: 19 or an amino acid        sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:        19, and        -   a second ISVD comprises a CDR1 that is the amino acid            sequence of SEQ ID NO: 11 or an amino acid sequence with 2            or 1 amino acid difference(s) with SEQ ID NO: 11, a CDR2            that is the amino acid sequence of SEQ ID NO: 16 or an amino            acid sequence with 2 or 1 amino acid difference(s) with SEQ            ID NO: 16, and a CDR3 that is the amino acid sequence of SEQ            ID NO: 21 or an amino acid sequence with 2 or 1 amino acid            difference(s) with SEQ ID NO: 21.

In one embodiment of this aspect of the present technology, the ISVDsare linked via one or more peptidic linkers. In one embodiment, the twoISVDs specifically bind human TSLP.

In a further embodiment, the monospecific-bivalent polypeptide comprisesor consists of two ISVDs that specifically bind to TSLP, wherein:

-   -   a) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 11, a CDR2 that is the amino acid        sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21, and the second ISVD comprises a CDR1        that is the amino acid sequence of SEQ ID NO: 9, a CDR2 that is        the amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 19, or    -   b) the first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence        of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 19, and the second ISVD comprises a CDR1 that is the        amino acid sequence of SEQ ID NO:11, a CDR2 that is the amino        acid sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21.

In a further embodiment of this aspect of the present technology, themonospecific-bivalent polypeptide comprises or consists of two ISVDsthat specifically bind to TSLP, wherein:

-   -   a) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 6 and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 4, or    -   b) the amino acid sequence of the first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 4 and the        second ISVD comprises a sequence identity of more than 90%        identity with SEQ ID NO: 6.

In one embodiment, the monospecific-bivalent polypeptide comprises orconsists of two ISVDs that specifically bind to TSLP, wherein:

-   -   a) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 6 and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 4; or    -   b) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 4 and the second ISVD comprises the amino acid sequence of        SEQ ID NO: 6.

The terms “first ISVD” and “second ISVD” in this regard only indicatethe relative position of the specifically recited ISVDs binding toIL-13/TSLP to each other, wherein the numbering is started from theN-terminus of the polypeptide of the present technology. The “firstISVD” is thus closer to the N-terminus than the “second ISVD”.Accordingly, the “second ISVD” is thus closer to the C-terminus than the“first ISVD”. Since the numbering is thus not absolute and onlyindicates the relative position of the two ISVDs it does not exclude thepossibility that additional binding units/building blocks such as ISVDsbinding to IL-13 and TSLP, respectively, can be present in thepolypeptide. Moreover, it does not exclude the possibility that otherbinding units/building blocks such as ISVDs can be placed in between.For instance, as described further below (see in particular, section“multispecific-multivalent polypeptides” and 5.4 “(In vivo) half-lifeextension”), the polypeptide can further comprise another ISVD bindingto human serum albumin that can even be located between the “first ISVD”and “second ISVD” (such a construct is then referred to as multispecificas described in the subsequent section).

In one embodiment, the (at least two) ISVDs of themonospecific-multivalent polypeptides, in particular of the abovedescribed monospecific-bivalent polypeptides, are linked via peptidiclinkers. The use of peptidic linkers to connect two or more(poly)peptides is well known in the art. Exemplary peptidic linkers thatcan be used with the monospecific-multivalent polypeptides, inparticular with the above described monospecific-bivalent polypeptidesare shown in Table A-5. One often used class of peptidic linkers isknown as the “Gly-Ser” or “GS” linkers. These are linkers thatessentially consist of glycine (G) and serine (S) residues, and usuallycomprise one or more repeats of a peptide motif such as the GGGGS (SEQID NO: 73) motif (for example, comprising the formula(Gly-Gly-Gly-Gly-Ser)_(n) in which n may be 1, 2, 3, 4, 5, 6, 7 ormore). Some often used examples of such GS linkers are 9GS linkers(GGGGSGGGS, SEQ ID NO: 76) 15GS linkers (n=3) and 35GS linkers (n=7).Reference is for example made to Chen et al., Adv. Drug Deliv. Rev. 2013Oct. 15; 65(10): 1357-1369; and Klein et al., Protein Eng. Des. Sel.(2014) 27 (10): 325-330. In one embodiment of the present technology,the ISVDs of the monospecific-multivalent polypeptides, in particularthe monospecific-bivalent polypeptides of the present technology arelinked via a linker set forth in Table A-5. In one embodiment, the (atleast) two ISVDs are linked via a 35GS linker(s).

Accordingly, in one embodiment, the monospecific-bivalent polypeptidecomprises or consists of:

-   -   a) the amino acid sequence of SEQ ID NO: 148,    -   b) the amino acid sequence of SEQ ID NO: 149,    -   c) the amino acid sequence of SEQ ID NO: 150,    -   d) the amino acid sequence of SEQ ID NO: 151,    -   e) the amino acid sequence of SEQ ID NO: 152,    -   f) the amino acid sequence of SEQ ID NO: 153,    -   g) the amino acid sequence of SEQ ID NO: 154,    -   h) the amino acid sequence of SEQ ID NO: 155,    -   i) the amino acid sequence of SEQ ID NO: 156,    -   j) the amino acid sequence of SEQ ID NO: 157,    -   k) the amino acid sequence of SEQ ID NO: 158, or    -   l) the amino acid sequence of SEQ ID NO: 159.

Multispecific-Multivalent Polypeptides

In a further aspect, the polypeptide of the present technology is atleast bispecific, but can also be e.g., trispecific, tetraspecific,pentaspecific, etc. Moreover, the polypeptide is at least bivalent, butcan also be e.g., trivalent, tetravalent, pentavalent, hexavalent, etc.

The terms “bispecific”, “trispecific”, “tetraspecific”, “pentaspecific”,etc., all fall under the term “multispecific” and refer to binding totwo, three, four, five, etc., different target molecules, respectively.

The terms “bivalent”, “trivalent”, “tetravalent”, “pentavalent”,“hexavalent”, etc. all fall under the term “multivalent” and indicatethe presence of two, three, four, five, six, etc., bindingunits/building blocks, respectively, such as ISVDs.

For example, the polypeptide may be bispecific-tetravalent, such as apolypeptide comprising or consisting of at least four ISVDs, wherein atleast two ISVD specifically bind to IL-13 and at least two ISVDsspecifically bind to TSLP. In one embodiment, IL-13 and TSLP are humanIL-13 and human TSLP. In another example, the polypeptide may betrispecific-pentavalent, such as a polypeptide comprising or consistingof five ISVDs, wherein two ISVDs specifically bind to human IL-13, twoISVDs specifically bind to human TSLP and one ISVD binds to human serumalbumin. Such a polypeptide may at the same time be biparatopic, forexample if two ISVDs bind two different epitopes on human IL-13 or humanTSLP. The term “biparatopic” refers to binding to two different parts(e.g., epitopes) of the same target molecule. In one embodiment, thetrispecific-pentavalent polypeptide of the present technology is e.g.,ISVD construct F027400161, comprising two ISVDs specifically binding tohuman IL-13, two ISVDs specifically binding to human TSLP, one ISVDbinding to human serum albumin, and which is biparatopic for bothbinding to IL-13 and TSLP.

In one embodiment, the multispecific-multivalent polypeptide comprisesor consists of at least four ISVDs, wherein each of said ISVDs comprisesthree complementarity determining regions (CDR1 to CDR3, respectively),wherein the at least four ISVDs are optionally linked via one or morepeptidic linkers, and wherein:

-   -   a) a first ISVD that specifically binds IL-13 and comprises a        CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an amino        acid sequence with 2 or 1 amino acid difference(s) with SEQ ID        NO: 7, a CDR2 that is the amino acid sequence of SEQ ID NO: 12        or an amino acid sequence with 2 or 1 amino acid difference(s)        with SEQ ID NO: 12, and a CDR3 that is the amino acid sequence        of SEQ ID NO: 17 or an amino acid sequence with 2 or 1 amino        acid difference(s) with SEQ ID NO: 17;    -   b) a second ISVD that specifically binds IL-13 and comprises a        CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an amino        acid sequence with 2 or 1 amino acid difference(s) with SEQ ID        NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13        or an amino acid sequence with 2 or 1 amino acid difference(s)        with SEQ ID NO: 13, and a CDR3 that is the amino acid sequence        of SEQ ID NO: 18 or an amino acid sequence with 2 or 1 amino        acid difference(s) with SEQ ID NO: 18;    -   c) a third ISVD that specifically binds TSLP and comprises a        CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an amino        acid sequence with 2 or 1 amino acid difference(s) with SEQ ID        NO: 9, a CDR2 that is the amino acid sequence of SEQ ID NO: 14        or an amino acid sequence with 2 or 1 amino acid difference(s)        with SEQ ID NO: 14, and a CDR3 that is the amino acid sequence        of SEQ ID NO: 19 or an amino acid sequence with 2 or 1 amino        acid difference(s) with SEQ ID NO: 19, and    -   d) a fourth ISVD that specifically binds TSLP and comprises a        CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 11, a CDR2 that is the amino acid sequence of SEQ ID        NO: 16 or an amino acid sequence with 2 or 1 amino acid        difference(s) with SEQ ID NO: 16, and a CDR3 that is the amino        acid sequence of SEQ ID NO: 21 or an amino acid sequence with 2        or 1 amino acid difference(s) with SEQ ID NO: 21.

In one embodiment, the IL-13 and TSLP bound by said polypeptide is humanIL-13 and human TSLP, respectively.

In a further embodiment of the multispecific-multivalent polypeptide:

-   -   a) said first ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 7, a CDR2 that is the amino acid sequence        of SEQ ID NO: 12 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 17;    -   b) said second ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence        of SEQ ID NO: 13 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 18;    -   c) said third ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 9, a CDR2 that is the amino acid sequence        of SEQ ID NO: 14 and a CDR3 that is the amino acid sequence of        SEQ ID NO: 19; and    -   d) said fourth ISVD comprises a CDR1 that is the amino acid        sequence of SEQ ID NO: 11, a CDR2 that is the amino acid        sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21.

In a further aspect of the multispecific-multivalent polypeptide:

-   -   a) the amino acid sequence of said first ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 2;    -   b) the amino acid sequence of said second ISVD comprises a        sequence identity of more than 90% with SEQ ID NO: 3;    -   c) the amino acid sequence of said third ISVD comprises a        sequence identity of more than 90% identity with SEQ ID NO: 4;        and    -   d) the amino acid sequence of said fourth ISVD comprises a        sequence identity of more than 90% identity with SEQ ID NO: 6.

In one embodiment of the multispecific-multivalent polypeptide:

-   -   a) the first ISVD comprises the amino acid sequence of SEQ ID        NO: 2;    -   b) the second ISVD comprises the amino acid sequence of SEQ ID        NO: 3;    -   c) the third ISVD comprises the amino acid sequence of SEQ ID        NO: 4; and    -   d) the fourth ISVD comprises the amino acid sequence of SEQ ID        NO: 6.

The terms “first ISVD”, “second ISVD”, “third ISVD”, etc., in thisregard only indicate the relative position of the ISVDs to each other,wherein the numbering is started from the N-terminus of the polypeptideof the present technology. The “first ISVD” is thus closer to theN-terminus than the “second ISVD”, whereas the “second ISVD” is closerto the N-terminus than the “third ISVD”. Accordingly, the ISVDarrangement is inverse when considered from the C-terminus. Since thenumbering is not absolute and only indicates the relative position ofthe at least four ISVDs it is not excluded that other bindingunits/building blocks such as additional ISVDs binding to IL-13 or TSLP,or ISVDs binding to another target may be present in the polypeptide.Moreover, it does not exclude the possibility that other bindingunits/building blocks such as ISVDs can be placed in between. Forinstance, as described further below (see in particular, section 5.4“(In vivo) half-life extension” below), the polypeptide can furthercomprise another ISVD binding to human serum albumin that can even belocated between e.g. the “third ISVD” and “fourth ISVD”.

In another aspect the present technology provides a bispecific-bivalentpolypeptide comprising an ISVD that specifically binds to IL-13 or TSLPas described in detail for the monospecific-monovalent polypeptidesabove (section 5.1; “Monospecific-monovalent polypeptides”) and an ISVDbinding to human serum albumin as described in detail below (section5.4; “(In vivo) half-life extension”).

In another aspect the present technology provides a bispecific-trivalentpolypeptide comprising the monospecific-bivalent polypeptides above(section 5.1; “monospecific-bivalent polypeptides”) and an ISVD bindingto human serum albumin as described in detail below (section 5.4; “(Invivo) half-life extension”).

The components, such as the ISVDs, of said multispecific-multivalentpolypeptide may be linked to each other by one or more suitable linkers,such as peptidic linkers.

The use of linkers to connect two or more (poly)peptides is well knownin the art. Exemplary peptidic linkers are shown in Table A-5. One oftenused class of peptidic linker are known as the “Gly-Ser” or “GS”linkers. These are linkers that essentially consist of glycine (G) andserine (S) residues, and usually comprise one or more repeats of apeptide motif such as the GGGGS (SEQ ID NO: 73) motif (for example,comprising the formula (Gly-Gly-Gly-Gly-Ser), in which n may be 1, 2, 3,4, 5, 6, 7 or more). Some often used examples of such GS linkers are 9GSlinkers (GGGGSGGGS, SEQ ID NO: 76) 15G5 linkers (n=3) (SEQ ID NO: 78)and 35GS linkers (n=7) (SEQ ID NO: 83). Reference is for example made toChen et al., Adv. Drug Deliv. Rev. 2013 Oct. 15; 65(10): 1357-1369; andKlein et al., Protein Eng. Des. Sel. (2014) 27 (10): 325-330. In thepolypeptide(s) of the present technology, the 9GS (SEQ ID NO: 76) and35GS (SEQ ID NO: 83) linkers are used to link the components of thepolypeptide to each other.

In one aspect of the multispecific-multivalent polypeptide of thepresent technology, the polypeptide comprising or consisting of at leastfour ISVDs, comprises the at least two ISVDs specifically binding toIL-13 and at least two ISVDs specifically binding to TSLP. In thisaspect of the present technology, the at least two ISVDs binding toIL-13 are linked via a 35GS linker, whereas the at least two ISVDsspecifically binding to TSLP are linked via a 9GS linker. In oneembodiment, the at least two ISVDs specifically binding to TSLP areseparated by an ISVD binding to albumin (9GS-Alb-9GS) (as described insection 5.4 “(In vivo) half-life extension” below). The inventorssurprisingly found that such a configuration can increase the productionyield of the polypeptide.

Accordingly, in one embodiment, the polypeptide comprises or consists ofthe following, in the order starting from the N-terminus of thepolypeptide: a first ISVD specifically binding to IL-13, a second ISVDspecifically binding to IL-13, a first ISVD specifically binding toTSLP, an optional binding unit providing the polypeptide with increasedhalf-life as defined herein, and a second ISVD specifically binding toTSLP. In one embodiment, the binding unit providing the polypeptide withincreased half-life is an ISVD.

In a further embodiment, the polypeptide comprises or consists of thefollowing, in the order starting from the N-terminus of the polypeptide:an ISVD specifically binding to IL-13, a linker, a second ISVDspecifically binding to IL-13, a linker, a first ISVD specificallybinding to TSLP, a linker, an ISVD binding to human serum albumin, alinker, and a second ISVD specifically binding to TSLP. In one specificembodiment, the linker between the two ISVDs binding to IL-13 is a 35GSlinker, whereas the other linkers are 9GS linkers.

Such configurations of the polypeptide can provide for increasedproduction yield, good CMC characteristics, such as sufficientsolubility and biophysical stability, strong potencies with regard tomodulation of a type 2 immune response as well as low binding topre-existing antibodies.

In one embodiment, the multispecific-multivalent polypeptide of thepresent technology exhibits reduced binding by pre-existing antibodiesin human serum. To this end, in one embodiment, the polypeptidecomprises a valine (V) at amino acid position 11 and a leucine (L) atamino acid position 89 (according to Kabat numbering) in at least oneISVD. In one embodiment, the polypeptide comprises a valine (V) at aminoacid position 11 and a leucine (L) at amino acid position 89 (accordingto Kabat numbering) in each ISVD. In another embodiment, the polypeptidecomprises an extension of 1 to 5 (naturally occurring) amino acids, suchas a single alanine (A) extension, at the C-terminus of the C-terminalISVD. The C-terminus of an ISVD is normally VTVSS (SEQ ID NO: 138). Inanother embodiment, the polypeptide comprises a lysine (K) or glutamine(Q) at position 110 (according to Kabat numbering) in at least one ISVD.In another embodiment, the ISVD comprises a lysine (K) or glutamine (Q)at position 112 (according to Kabat numbering) in at least on ISVD. Inthese embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 139),VQVSS (SEQ ID NO: 140), VTVKS (SEQ ID NO:166), VTVQS (SEQ ID NO:167),VKVKS (SEQ ID NO:168), VKVQS (SEQ ID NO:169), VQVKS (SEQ ID NO:170), orVQVQS (SEQ ID NO:171) such that after addition of a single alanine theC-terminus of the polypeptide for example comprises the sequence VTVSSA(SEQ ID NO: 141), VKVSSA (SEQ ID NO: 142), VQVSSA (SEQ ID NO: 143),VTVKSA (SEQ ID NO:172), VTVQSA (SEQ ID NO:173), VKVKSA (SEQ ID NO:174),VKVQSA (SEQ ID NO:175), VQVKSA (SEQ ID NO:176), or VQVQSA (SEQ IDNO:177). In one embodiment, the C-terminus comprises VTVSSA (SEQ ID NO:141). In another embodiment, the polypeptide comprises a valine (V) atamino acid position 11 and a leucine (L) at amino acid position 89(according to Kabat numbering) in each ISVD, optionally a lysine (K) orglutamine (Q) at position 110 (according to Kabat numbering) in at leastone ISVD and comprises an extension of 1 to 5 (naturally occurring)amino acids, such as a single alanine (A) extension, at the C-terminusof the C-terminal ISVD (such that the C-terminus of the polypeptide forexample has the sequence VTVSSA (SEQ ID NO: 141), VKVSSA (SEQ ID NO:142) or VQVSSA (SEQ ID NO: 143), such as VTVSSA (SEQ ID NO: 141)). Seee.g. WO2012/175741 and WO2015/173325 for further information in thisregard.

In one embodiment, the multispecific-multivalent polypeptide of thepresent technology comprises or consists of an amino acid sequencecomprising a sequence identity of more than 90%, such as more than 95%or more than 99%, with SEQ ID NO: 1, wherein the CDRs of the five ISVDsare as defined in items A to E (or A′ to E′ if using the Kabatdefinition) set forth in sections “5.2 Immunoglobulin single variabledomains” and “5.4 (In vivo) half-life extension” below, respectively,wherein in particular:

-   -   the first ISVD specifically binding to IL-13 has a CDR1 that is        the amino acid sequence of SEQ ID NO: 7, a CDR2 that is the        amino acid sequence of SEQ ID NO: 12 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 17;    -   the second ISVD specifically binding to IL-13 has a CDR1 that is        the amino acid sequence of SEQ ID NO: 8, a CDR2 that is the        amino acid sequence of SEQ ID NO: 13 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 18;    -   the third ISVD specifically binding to TSLP has a CDR1 that is        the amino acid sequence of SEQ ID NO: 9, a CDR2 that is the        amino acid sequence of SEQ ID NO: 14 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 19;    -   the fourth ISVD specifically binding to TSLP has a CDR1 that is        the amino acid sequence of SEQ ID NO: 11, a CDR2 that is the        amino acid sequence of SEQ ID NO: 16 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 21; and    -   the ISVD binding to human serum albumin has a CDR1 that is the        amino acid sequence of SEQ ID NO: 10, a CDR2 that is the amino        acid sequence of SEQ ID NO: 15 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 20,

or alternatively if using the Kabat definition:

-   -   the first ISVD specifically binding to IL-13 has a CDR1 that is        the amino acid sequence of SEQ ID NO: 37, a CDR2 that is the        amino acid sequence of SEQ ID NO: 42 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 17;    -   the second ISVD specifically binding to IL-13 has a CDR1 that is        the amino acid sequence of SEQ ID NO: 38, a CDR2 that is the        amino acid sequence of SEQ ID NO: 43 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 18;    -   the third ISVD specifically binding to TSLP has a CDR1 that is        the amino acid sequence of SEQ ID NO: 39, a CDR2 that is the        amino acid sequence of SEQ ID NO: 44 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 19;    -   the fourth ISVD specifically binding to TSLP has a CDR1 that is        the amino acid sequence of SEQ ID NO: 41, a CDR2 that is the        amino acid sequence of SEQ ID NO: 46 and a CDR3 that is the        amino acid sequence of SEQ ID NO: 21; and    -   the ISVD binding to human serum albumin has a CDR1 that is the        amino acid sequence of SEQ ID NO: 40, a CDR2 that is the amino        acid sequence of SEQ ID NO: 45 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 20.

In another embodiment, the polypeptide comprises or consists of theamino acid sequence of SEQ ID NO: 1. In another embodiment, thepolypeptide consists of the amino acid sequence of SEQ ID NO: 1.

In one embodiment, the polypeptide of the present technology has atleast half the binding affinity, or at least the same binding affinity,to human IL-13 and to human TSLP as compared to a polypeptide consistingof the amino acid of SEQ ID NO: 1 wherein the binding affinity ismeasured using the same method, such as Surface Plasmon Resonance (SPR).

5.2 Immunoglobulin Single Variable Domains

The term “immunoglobulin single variable domain” (ISVD), interchangeablyused with “single variable domain”, defines immunoglobulin moleculeswherein the antigen binding site is present on, and formed by, a singleimmunoglobulin domain. This sets ISVDs apart from “conventional”immunoglobulins (e.g. monoclonal antibodies) or their fragments (such asFab, Fab′, F(ab′)₂, scFv, di-scFv), wherein two immunoglobulin domains,in particular two variable domains, interact to form an antigen bindingsite. Typically, in conventional immunoglobulins, a heavy chain variabledomain (V_(H)) and a light chain variable domain (V_(L)) interact toform an antigen binding site. In this case, the complementaritydetermining regions (CDRs) of both V_(H) and V_(L) will contribute tothe antigen binding site, i.e. a total of 6 CDRs will be involved inantigen binding site formation.

In view of the above definition, the antigen-binding domain of aconventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgEmolecule; known in the art) or of a Fab fragment, a F(ab′)₂ fragment, anFv fragment such as a disulphide linked Fv or a scFv fragment, or adiabody (all known in the art) derived from such conventional 4-chainantibody, would normally not be regarded as an ISVD, as, in these cases,binding to the respective epitope of an antigen would normally not occurby one (single) immunoglobulin domain but by a pair of (associating)immunoglobulin domains such as light and heavy chain variable domains,i.e., by a V_(H)-V_(L) pair of immunoglobulin domains, which jointlybind to an epitope of the respective antigen.

In contrast, ISVDs are capable of specifically binding to an epitope ofthe antigen without pairing with an additional immunoglobulin variabledomain. The binding site of an ISVD is formed by a single V_(H), asingle V_(HH) or single V_(L) domain.

As such, the single variable domain may be a light chain variable domainsequence (e.g., a V_(L)-sequence) or a suitable fragment thereof; or aheavy chain variable domain sequence (e.g., a V_(H)-sequence or V_(HH)sequence) or a suitable fragment thereof; as long as it is capable offorming a single antigen binding unit (i.e., a functional antigenbinding unit that essentially consists of the single variable domain,such that the single antigen binding domain does not need to interactwith another variable domain to form a functional antigen binding unit).

An ISVD can for example be a heavy chain ISVD, such as a V_(H), V_(HH),including a camelized V_(H) or humanized V_(HH). In one embodiment, itis a V_(HH), including a camelized V_(H) or humanized V_(HH). Heavychain ISVDs can be derived from a conventional four-chain antibody orfrom a heavy chain antibody.

For example, the ISVD may be a single domain antibody (or an amino acidsequence that is suitable for use as a single domain antibody), a “dAb”or dAb (or an amino acid sequence that is suitable for use as a dAb) ora Nanobody® (as defined herein, and including but not limited to aV_(HH)); other single variable domains, or any suitable fragment of anyone thereof.

In particular, the ISVD may be a Nanobody® (such as a V_(HH), includinga humanized V_(HH) or camelized V_(H)) or a suitable fragment thereof.Nanobody®, Nanobodies® and Nanoclone® are registered trademarks ofAblynx N.V.

“V_(HH) domains”, also known as V_(HH)5, V_(HH) antibody fragments, andV_(HH) antibodies, have originally been described as the antigen bindingimmunoglobulin variable domain of “heavy chain antibodies” (i.e., of“antibodies devoid of light chains”; Hamers-Casterman et al. Nature 363:446-448, 1993). The term “V_(HH) domain” has been chosen in order todistinguish these variable domains from the heavy chain variable domainsthat are present in conventional 4-chain antibodies (which are referredto herein as “V_(H) domains”) and from the light chain variable domainsthat are present in conventional 4-chain antibodies (which are referredto herein as “V_(L) domains”). For a further description of V_(HH)'s,reference is made to the review article by Muyldermans (Reviews inMolecular Biotechnology 74: 277-302, 2001).

Typically, the generation of immunoglobulins involves the immunizationof experimental animals, fusion of immunoglobulin producing cells tocreate hybridomas and screening for the desired specificities.Alternatively, immunoglobulins can be generated by screening of naïve orsynthetic libraries e.g. by phage display.

The generation of immunoglobulin sequences, such as Nanobodies®, hasbeen described extensively in various publications, among which WO94/04678, Hamers-Casterman et al. 1993 and Muyldermans et al. 2001 canbe exemplified. In these methods, camelids are immunized with the targetantigen in order to induce an immune response against said targetantigen. The repertoire of Nanobodies obtained from said immunization isfurther screened for Nanobodies that bind the target antigen.

In these instances, the generation of antibodies requires purifiedantigen for immunization and/or screening. Antigens can be purified fromnatural sources, or in the course of recombinant production.

Immunization and/or screening for immunoglobulin sequences can beperformed using peptide fragments of such antigens.

The present technology may use immunoglobulin sequences of differentorigin, comprising mouse, rat, rabbit, donkey, human and camelidimmunoglobulin sequences. The present technology also includes fullyhuman, humanized or chimeric sequences. For example, the presenttechnology comprises camelid immunoglobulin sequences and humanizedcamelid immunoglobulin sequences, or camelized domain antibodies, e.g.camelized dAb as described by Ward et al (see for example WO 94/04678and Davies and Riechmann (1994 and 1996)). Moreover, the presenttechnology also uses fused immunoglobulin sequences, e.g. forming amultivalent and/or multispecific construct (for multivalent andmultispecific polypeptides containing one or more V_(HH) domains andtheir preparation, reference is also made to Conrath et al., J. Biol.Chem., Vol. 276, 10. 7346-7350, 2001, as well as to for example WO96/34103 and WO 99/23221), and immunoglobulin sequences comprising tagsor other functional moieties, e.g. toxins, labels, radiochemicals, etc.,which are derivable from the immunoglobulin sequences of the presenttechnology.

A “humanized V_(HH)” comprises an amino acid sequence that correspondsto the amino acid sequence of a naturally occurring V_(HH) domain, butthat has been “humanized”, i.e. by replacing one or more amino acidresidues in the amino acid sequence of said naturally occurring V_(HH)sequence (and in particular in the framework sequences) by one or moreof the amino acid residues that occur at the corresponding position(s)in a V_(H) domain from a conventional 4-chain antibody from a humanbeing (e.g. indicated above). This can be performed in a manner knownper se, which will be clear to the skilled person, for example on thebasis of the further description herein and the prior art (e.g. WO2008/020079). Again, it should be noted that such humanized V_(HH)s canbe obtained in any suitable manner known per se and thus are notstrictly limited to polypeptides that have been obtained using apolypeptide that comprises a naturally occurring V_(HH) domain as astarting material.

A “camelized V_(H)” comprises an amino acid sequence that corresponds tothe amino acid sequence of a naturally occurring V_(H) domain, but thathas been “camelized”, i.e. by replacing one or more amino acid residuesin the amino acid sequence of a naturally occurring V_(H) domain from aconventional 4-chain antibody by one or more of the amino acid residuesthat occur at the corresponding position(s) in a V_(HH) domain of aheavy chain antibody. This can be performed in a manner known per se,which will be clear to the skilled person, for example on the basis ofthe further description herein and the prior art (e.g. WO 2008/020079).Such “camelizing” substitutions are usually inserted at amino acidpositions that form and/or are present at the V_(H)—V_(L) interface,and/or at the so-called Camelidae hallmark residues, as defined herein(see for example WO 94/04678 and Davies and Riechmann (1994 and 1996),supra). In one embodiment, the V_(H) sequence that is used as a startingmaterial or starting point for generating or designing the camelizedV_(H) is a V_(H) sequence from a mammal, or the V_(H) sequence of ahuman being, such as a V_(H)3 sequence. However, it should be noted thatsuch camelized V_(H) can be obtained in any suitable manner known per seand thus are not strictly limited to polypeptides that have beenobtained using a polypeptide that comprises a naturally occurring V_(H)domain as a starting material.

The structure of an ISVD sequence can be considered to be comprised offour framework regions (“FRs”), which are referred to in the art andherein as “Framework region 1” (“FR1”); as “Framework region 2” (“FR2”);as “Framework region 3” (“FR3”); and as “Framework region 4” (“FR4”),respectively; which framework regions are interrupted by threecomplementary determining regions (“CDRs”), which are referred to in theart and herein as “Complementarity Determining Region 1” (“CDR1”); as“Complementarity Determining Region 2” (“CDR2”); and as “ComplementarityDetermining Region 3” (“CDR3”), respectively.

As further described in paragraph q) on pages 58 and 59 of WO 08/020079,the amino acid residues of an ISVD can be numbered according to thegeneral numbering for V_(H) domains given by Kabat et al. (“Sequence ofproteins of immunological interest”, US Public Health Services, NIHBethesda, Md., Publication No. 91), as applied to V_(HH) domains fromCamelids in the article of Riechmann and Muyldermans, 2000 (J. Immunol.Methods 240 (1-2): 185-195; see for example FIG. 2 of this publication).It should be noted that—as is well known in the art for V_(H) domainsand for V_(HH) domains—the total number of amino acid residues in eachof the CDRs may vary and may not correspond to the total number of aminoacid residues indicated by the Kabat numbering (that is, one or morepositions according to the Kabat numbering may not be occupied in theactual sequence, or the actual sequence may contain more amino acidresidues than the number allowed for by the Kabat numbering). This meansthat, generally, the numbering according to Kabat may or may notcorrespond to the actual numbering of the amino acid residues in theactual sequence. The total number of amino acid residues in a V_(H)domain and a V_(HH) domain will usually be in the range of from 110 to120, often between 112 and 115. It should however be noted that smallerand longer sequences may also be suitable for the purposes describedherein.

In the present application, unless indicated otherwise, CDR sequenceswere determined according to the AbM definition as described inKontermann and Dübel (Eds. 2010, Antibody Engineering, vol 2, SpringerVerlag Heidelberg Berlin, Martin, Chapter 3, pp. 33-51). According tothis method, FR1 comprises the amino acid residues at positions 1-25,CDR1 comprises the amino acid residues at positions 26-35, FR2 comprisesthe amino acids at positions 36-49, CDR2 comprises the amino acidresidues at positions 50-58, FR3 comprises the amino acid residues atpositions 59-94, CDR3 comprises the amino acid residues at positions95-102, and FR4 comprises the amino acid residues at positions 103-113.

Determination of CDR regions may also be done according to differentmethods. In the CDR determination according to Kabat, FR1 of an ISVDcomprises the amino acid residues at positions 1-30, CDR1 of an ISVDcomprises the amino acid residues at positions 31-35, FR2 of an ISVDcomprises the amino acids at positions 36-49, CDR2 of an ISVD comprisesthe amino acid residues at positions 50-65, FR3 of an ISVD comprises theamino acid residues at positions 66-94, CDR3 of an ISVD comprises theamino acid residues at positions 95-102, and FR4 of an ISVD comprisesthe amino acid residues at positions 103-113.

In such an immunoglobulin sequence, the framework sequences may be anysuitable framework sequences, and examples of suitable frameworksequences will be clear to the skilled person, for example on the basisthe standard handbooks and the further disclosure and prior artmentioned herein.

The framework sequences are a suitable combination of immunoglobulinframework sequences or framework sequences that have been derived fromimmunoglobulin framework sequences (for example, by humanization orcamelization). For example, the framework sequences may be frameworksequences derived from a light chain variable domain (e.g. aV_(L)-sequence) and/or from a heavy chain variable domain (e.g. aV_(H)-sequence or V_(HH) sequence). In one aspect, the frameworksequences are either framework sequences that have been derived from aV_(HH)-sequence (in which said framework sequences may optionally havebeen partially or fully humanized) or are conventional V_(H) sequencesthat have been camelized (as defined herein).

In particular, the framework sequences present in the ISVD sequence usedin the present technology may contain one or more of hallmark residues(as defined herein), such that the ISVD sequence is a Nanobody®, such asa V_(HH), including a humanized V_(HH) or camelized V_(H). Somenon-limiting examples of (suitable combinations of) such frameworksequences will become clear from the further disclosure herein.

Again, as generally described herein for the immunoglobulin sequences,it is also possible to use suitable fragments (or combinations offragments) of any of the foregoing, such as fragments that contain oneor more CDR sequences, suitably flanked by and/or linked via one or moreframework sequences (for example, in the same order as these CDR's andframework sequences may occur in the full-sized immunoglobulin sequencefrom which the fragment has been derived).

However, it should be noted that the present technology is not limitedas to the origin of the ISVD sequence (or of the nucleotide sequenceused to express it), nor as to the way that the ISVD sequence ornucleotide sequence is (or has been) generated or obtained. Thus, theISVD sequences may be naturally occurring sequences (from any suitablespecies) or synthetic or semi-synthetic sequences. In a specific butnon-limiting aspect, the ISVD sequence is a naturally occurring sequence(from any suitable species) or a synthetic or semi-synthetic sequence,including but not limited to “humanized” (as defined herein)immunoglobulin sequences (such as partially or fully humanized mouse orrabbit immunoglobulin sequences, and in particular partially or fullyhumanized V_(HH) sequences), “camelized” (as defined herein)immunoglobulin sequences, as well as immunoglobulin sequences that havebeen obtained by techniques such as affinity maturation (for example,starting from synthetic, random or naturally occurring immunoglobulinsequences), CDR grafting, veneering, combining fragments derived fromdifferent immunoglobulin sequences, PCR assembly using overlappingprimers, and similar techniques for engineering immunoglobulin sequenceswell known to the skilled person; or any suitable combination of any ofthe foregoing.

Similarly, nucleotide sequences may be naturally occurring nucleotidesequences or synthetic or semi-synthetic sequences, and may for examplebe sequences that are isolated by PCR from a suitable naturallyoccurring template (e.g. DNA or RNA isolated from a cell), nucleotidesequences that have been isolated from a library (and in particular, anexpression library), nucleotide sequences that have been prepared byintroducing mutations into a naturally occurring nucleotide sequence(using any suitable technique known per se, such as mismatch PCR),nucleotide sequence that have been prepared by PCR using overlappingprimers, or nucleotide sequences that have been prepared usingtechniques for DNA synthesis known per se.

As described above, an ISVD may be a Nanobody® or a suitable fragmentthereof. For a general description of Nanobodies, reference is made tothe further description below, as well as to the prior art cited herein.In this respect, it should however be noted that this description andthe prior art mainly described Nanobodies of the so-called “V_(H)3class” (i.e. Nanobodies with a high degree of sequence homology to humangermline sequences of the V_(H)3 class such as DP-47, DP-51 or DP-29).It should however be noted that the present technology in its broadestsense can generally use any type of Nanobody, and for example also usesthe Nanobodies belonging to the so-called “V_(H)4 class” (i.e.Nanobodies with a high degree of sequence homology to human germlinesequences of the V_(H)4 class such as DP-78), as for example describedin WO 2007/118670.

Generally, Nanobodies (in particular V_(HH) sequences, including(partially) humanized V_(HH) sequences and camelized V_(H) sequences)can be characterized by the presence of one or more “Hallmark residues”(as described herein) in one or more of the framework sequences (againas further described herein). Thus, generally, a Nanobody can be definedas an immunoglobulin sequence with the (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which one or more of the Hallmark residuesare as further defined herein.

In particular, a Nanobody can be an immunoglobulin sequence with the(general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which the framework sequences are as furtherdefined herein.

More in particular, a Nanobody can be an immunoglobulin sequence withthe (general) structure

-   -   FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4

in which FR1 to FR4 refer to framework regions 1 to 4, respectively, andin which CDR1 to CDR3 refer to the complementarity determining regions 1to 3, respectively, and in which:

one or more of the amino acid residues at positions 11, 37, 44, 45, 47,83, 84, 103, 104 and 108 according to the Kabat numbering are chosenfrom the Hallmark residues mentioned in Table A-0 below.

TABLE A-0 Hallmark Residues in Nanobodies Position Human V_(H)3 HallmarkResidues 11 L, V; predominantly L L, S, V, M, W, F, T, Q, E, A, R, G, K,Y, N, P, I; preferably L 37 V, I, F; usually V F⁽¹⁾, Y, V, L, A, H, S,I, W, C, N, G, D, T, P, preferably F⁽¹⁾ or Y 44⁽⁸⁾ G E⁽³⁾, Q⁽³⁾, G⁽²⁾,D, A, K, R, L, P, S, V, H, T, N, W, M, I; preferably G⁽²⁾, E⁽³⁾ or Q⁽³⁾;most preferably G⁽²⁾ or Q⁽³⁾. 45⁽⁸⁾ L L⁽²⁾, R⁽³⁾, P, H, F, G, Q, S, E,T, Y, C, I, D, V; preferably L⁽²⁾ or R⁽³⁾ 47⁽⁸⁾ W, Y F⁽¹⁾, L⁽¹⁾ or W⁽²⁾G, I, S, A, V, M, R, Y, E, P, T, C, H, K, Q, N, D; preferably W⁽²⁾, L⁽¹⁾or F⁽¹⁾ 83 R or K; usually R R, K⁽⁵⁾, T, E⁽⁵⁾, Q, N, S, I, V, G, M, L,A, D, Y, H; preferably K or R; most preferably K 84 A, T, D;predominantly A P⁽⁵⁾, S, H, L, A, V, I, T, F, D, R, Y, N, Q, G, E;preferably P 103 W W⁽⁴⁾, R⁽⁶⁾, G, S, K, A, M, Y, L, F, T, N, V, Q, P⁽⁶⁾,E, C; preferably W 104 G G, A, S, T, D, P, N, E, C, L; preferably G 108L, M or T; predominantly L Q, L⁽⁷⁾, R, P, E, K, S, T, M, A, H;preferably Q or L⁽⁷⁾ Notes: ⁽¹⁾In particular, but not exclusively, incombination with KERE or KQRE at positions 43-46. ⁽²⁾Usually as GLEW atpositions 44-47. ⁽³⁾Usually as KERE or KQRE at positions 43-46, e.g. asKEREL, KEREF, KQREL, KQREF, KEREG, KQREW or KQREG at positions 43-47.Alternatively, also sequences such as TERE (for example TEREL), TQRE(for example TQREL), KECE (for example KECEL or KECER), KQCE (forexample KQCEL), RERE (for example REREG), RQRE (for example RQREL, RQREFor RQREW), QERE (for example QEREG), QQRE, (for example QQREW, QQREL orQQREF), KGRE (for example KGREG), KDRE (for example KDREV) are possible.Some other possible, but less preferred sequences include for exampleDECKL and NVCEL. ⁽⁴⁾With both GLEW at positions 44-47 and KERE or KQREat positions 43-46. ⁽⁵⁾Often as KP or EP at positions 83-84 of naturallyoccurring V_(HH) domains. ⁽⁶⁾In particular, but not exclusively, incombination with GLEW at positions 44-47. ⁽⁷⁾With the proviso that whenpositions 44-47 are GLEW, position 108 is always Q in (non-humanized)V_(HH) sequences that also contain a W at 103. ⁽⁸⁾The GLEW group alsocontains GLEW-like sequences at positions 44-47, such as for exampleGVEW, EPEW, GLER, DQEW, DLEW, GIEW, ELEW, GPEW, EWLP, GPER, GLER andELEW.

The present technology inter alia uses ISVDs that can bind to IL-13 orTSLP. In the context of the present technology, “binding to” a certaintarget molecule has the usual meaning in the art as understood in thecontext of antibodies and their respective antigens.

The multispecific-multivalent polypeptide of the present technology maycomprise two or more ISVDs specifically binding to IL-13 and two or moreISVDs specifically binding to TSLP. For example, the polypeptide maycomprise two ISVDs that specifically bind to IL-13 and two ISVDs thatspecifically bind to TSLP.

In some embodiments, at least one ISVD can functionally block its targetmolecule. For example, targeting moieties can block the interactionbetween IL-13 and IL-13Rα1 (Interleukin 13 receptor, alpha 1) and/or theinteraction between IL-13/IL-13Rα1 complex and IL-4Rα (alphainterleukin-4 receptor), or can block the interaction between TSLP andTSLPR (TSLP receptor) and/or TSLP/TSLPR complex and IL-7Rα(Interleukin-7 receptor subunit alpha). Accordingly, in one embodiment,the polypeptide of the present technology comprises at least two ISVDsthat specifically bind to IL-13 and functionally block its interactionwith IL-13Rα1 and/or the interaction between IL-13/IL-13Rα1 complex andIL-4Rα, and two ISVDs that specifically bind to TSLP and functionallyblock its interaction with TSLPR and/or the interaction betweenTSLP/TSLPR complex and IL-7Ra.

The ISVDs used in the present technology form part of a polypeptide ofthe present technology, which comprises or consists of at least fourISVDs, such that the polypeptide can specifically bind to IL-13 andTSLP.

Accordingly, the target molecules of the at least four ISVDs as used inthe polypeptide of the present technology are IL-13 and TSLP. Examplesare mammalian IL-13 and TSLP. Besides human IL-13 (Uniprot accessionP35225) and human TSLP (Uniprot accession Q969D9), the versions fromother species are also amenable to the present technology, for exampleIL-13 and TSLP from mice, rats, rabbits, cats, dogs, goats, sheep,horses, pigs, non-human primates, such as cynomolgus monkeys (alsoreferred to herein as “cyno”), or camelids, such as llama or alpaca.

Specific examples of ISVDs specifically binding to IL-13 that can beused in the present technology are as described in the following items Aand B:

A. An ISVD that specifically binds to human IL-13 and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 7;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 12; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 17.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 7, a CDR2 that is the amino acid        sequence of SEQ ID NO: 12 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 17.

B. An ISVD that specifically binds to human IL-13 and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 8 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 8;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 13 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 13; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 18.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 8, a CDR2 that is the amino acid        sequence of SEQ ID NO: 13 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 18.

Examples of such an ISVD that specifically binds to human IL-13 have oneor more, or all, framework regions as indicated for construct 4B02 or4B06, respectively, in Table A-2 (in addition to the CDRs as defined inthe preceding items A and B, respectively). In one embodiment, it is anISVD comprising or consisting of the full amino acid sequence ofconstruct 4B02 or construct 4B06 (SEQ ID NOs: 2 and 3, respectively; seeTable A-1 and A-2).

In another embodiment, the amino acid sequence of the ISVD(s)specifically binding to human IL-13 may have a sequence identity of morethan 90%, such as more than 95% or more than 99%, with SEQ ID NO: 2 or 3respectively, wherein the CDRs are as defined in the preceding item A orB, respectively. In one embodiment, the ISVD specifically binding toIL-13 comprises or consists of the amino acid sequence of SEQ ID NO: 2or 3.

When such an ISVD binding to IL-13 has 2 or 1 amino acid difference inat least one CDR relative to a corresponding reference CDR sequence(item A or B above), the ISVD has at least half the binding affinity, orat least the same binding affinity to human IL-13 as the construct 4B02or 4B06 set forth in SEQ ID NO: 2 or 3, respectively, wherein thebinding affinity is measured using the same method, such as SPR.

Specific examples of ISVDs specifically binding to TSLP that can be usedin the present technology are as described in the following items C andD:

C. An ISVD that specifically binds to human TSLP and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 9 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 9;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 14 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 14; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 19.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 9, a CDR2 that is the amino acid        sequence of SEQ ID NO: 14 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 19.

D. An ISVD that specifically binds to human TSLP and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 11 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 11;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 16 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 16; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 21.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 11, a CDR2 that is the amino acid        sequence of SEQ ID NO: 16 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21.

Examples of such an ISVD that specifically binds to human TSLP have oneor more, or all, framework regions as indicated for construct 501A02 and529F10, respectively, in Table A-2 (in addition to the CDRs as definedin the preceding items C and D). In one embodiment, it is an ISVDcomprising or consisting of the full amino acid sequence of construct501A02 or 529F10 (SEQ ID NOs: 4 or 6, see Table A-1 and A-2).

In another embodiment, the amino acid sequence of an ISVD(s)specifically binding to human TSLP may have a sequence identity of morethan 90%, such as more than 95% or more than 99%, with SEQ ID NO: 4 or6, respectively, wherein the CDRs are as defined in the preceding item Cor D. In one embodiment, the ISVD binding to human TSLP comprises orconsists of the amino acid sequence of SEQ ID NOs: 4 or 6.

When such an ISVD binding to human TSLP has 2 or 1 amino acid differencein at least one CDR relative to a corresponding reference CDR sequence(item C or D above), the ISVD has at least half the binding affinity, orat least the same binding affinity to human TSLP as construct 501A02 or529F10 set forth in SEQ ID NO: 4 and 6, respectively, wherein thebinding affinity is measured using the same method, such as SPR.

In an embodiment, each of the ISVDs as defined under items A to D aboveis comprised in the polypeptide of the present technology.

Such a polypeptide of the present technology comprising each of theISVDs as defined under items A to D above has at least half the bindingaffinity, or at least the same binding affinity, to human IL-13 and tohuman TSLP as a polypeptide consisting of the amino acid of SEQ ID NO:1, wherein the binding affinity is measured using the same method, suchas SPR.

The SEQ ID NOs referred to in the above items A to D and item E below(see section 5.4 “(In vivo) half-life extension”) are based on the CDRdefinition according to the AbM definition (see Table A-2). It is notedthat the SEQ ID NOs defining the same CDRs according to the Kabatdefinition (see Table A-2.1) can likewise be used in the above items Ato D and item E below (see section 5.4 “(In vivo) half-life extension”).

Accordingly, the specific examples of ISVDs specifically binding toIL-13 or TSLP that can be used in the present technology are asdescribed above using the AbM definition can be also described using theKabat definition as set forth in items A′ to D′ below:

A′. An ISVD that specifically binds to human IL-13 and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 37 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 37;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 42 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 42; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 17.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 37, a CDR2 that is the amino acid        sequence of SEQ ID NO: 42 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 17.

B′. An ISVD that specifically binds to human IL-13 and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 38 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 38;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 43 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 43; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 18.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 38, a CDR2 that is the amino acid        sequence of SEQ ID NO: 43 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 18.

Examples of such an ISVD(s) that specifically binds to human IL-13 haveone or more, or all, framework regions as indicated for construct 4B02or 4B06, respectively, in Table A-2-1 (in addition to the CDRs asdefined in the preceding items A′ and B′, respectively). In oneembodiment it is an ISVD comprising or consisting of the full amino acidsequence of construct 4B02 or construct 4B06 (SEQ ID NOs: 2 and 3,respectively; see Table A-1 and A-2-1).

C′. An ISVD that specifically binds to human TSLP and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 39 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 39;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 44 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 44; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 19 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 19.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 39, a CDR2 that is the amino acid        sequence of SEQ ID NO: 44 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 19.

D′. An ISVD that specifically binds to human TSLP and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 41 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 41;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 46 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 46; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 21.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 41, a CDR2 that is the amino acid        sequence of SEQ ID NO: 46 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 21.

Examples of such an ISVD(s) that specifically binds to human TSLP haveone or more, or all, framework regions as indicated for construct 501A02and 529F10, respectively, in Table A-2-1 (in addition to the CDRs asdefined in the preceding items C′ and D′). In one embodiment, it is anISVD comprising or consisting of the full amino acid sequence ofconstruct 501A02 or 529F10 (SEQ ID NOs: 4 or 6, see Table A-1 andA-2-1).

The percentage of “sequence identity” between a first amino acidsequence and a second amino acid sequence may be calculated by dividing[the number of amino acid residues in the first amino acid sequence thatare identical to the amino acid residues at the corresponding positionsin the second amino acid sequence] by [the total number of amino acidresidues in the first amino acid sequence] and multiplying by [100%], inwhich each deletion, insertion, substitution or addition of an aminoacid residue in the second amino acid sequence—compared to the firstamino acid sequence—is considered as a difference at a single amino acidresidue (i.e. at a single position).

Usually, for the purpose of determining the percentage of “sequenceidentity” between two amino acid sequences in accordance with thecalculation method outlined hereinabove, the amino acid sequence withthe greatest number of amino acid residues will be taken as the “first”amino acid sequence, and the other amino acid sequence will be taken asthe “second” amino acid sequence.

An “amino acid difference” as used herein refers to a deletion,insertion or substitution of a single amino acid residue vis-à-vis areference sequence. In one embodiment, an “amino acid difference” is asubstitution.

In one embodiment, amino acid substitutions are conservativesubstitutions. Such conservative substitutions are substitutions inwhich one amino acid within the following groups (a)-(e) is substitutedby another amino acid residue within the same group: (a) smallaliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro andGly; (b) polar, negatively charged residues and their (uncharged)amides: Asp, Asn, Glu and Gln; (c) polar, positively charged residues:His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, Ile,Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

In one embodiment, conservative substitutions are as follows: Ala intoGly or into Ser; Arg into Lys; Asn into Gln or into His; Asp into Glu;Cys into Ser; Gln into Asn; Glu into Asp; Gly into Ala or into Pro; Hisinto Asn or into Gln; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gln or into Glu; Met into Leu, into Tyr or intoIle; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into Ile or into Leu.

5.3 Specificity

The terms “specificity”, “binding specifically” or “specific binding”refer to the number of different target molecules, such as antigens,from the same organism to which a particular binding unit, such as anISVD, can bind with sufficiently high affinity (see below).“Specificity”, “binding specifically” or “specific binding” are usedinterchangeably herein with “selectivity”, “binding selectively” or“selective binding”. Binding units, such as ISVDs, specifically bind totheir designated targets.

The specificity/selectivity of a binding unit can be determined based onaffinity. The affinity denotes the strength or stability of a molecularinteraction. The affinity is commonly given as by the KD, ordissociation constant, which has units of mol/liter (or M). The affinitycan also be expressed as an association constant, KA, which equals 1/KDand has units of (mol/liter)⁻¹ (or M⁻¹).

The affinity is a measure for the binding strength between a moiety anda binding site on the target molecule: the lower the value of the KD,the stronger the binding strength between a target molecule and atargeting moiety.

Typically, binding units used in the present technology (such as ISVDs)will bind to their targets with a dissociation constant (KD) of 10⁻⁵ to10⁻¹² moles/liter or less, or 10⁻⁷ to 10⁻¹² moles/liter or less, or 10⁻⁸to 10⁻¹² moles/liter (i.e. with an association constant (KA) of 10⁵ to10¹² liter/moles or more, or 10⁷ to 10¹² liter/moles or more, or 10⁸ to10¹² liter/moles).

Any KD value greater than 10⁻⁴ mol/liter (or any KA value lower than 10⁴liters/mol) is generally considered to indicate non-specific binding.

The KD for biological interactions, such as the binding ofimmunoglobulin sequences to an antigen, which are considered specificare typically in the range of 10⁻⁵ moles/liter (10000 nM or 10 μM) to10⁻¹² moles/liter (0.001 nM or 1 pM) or less.

Accordingly, specific/selective binding may mean that—using the samemeasurement method, e.g. SPR—a binding unit (or polypeptide comprisingthe same) binds to IL13 and/or TSLP with a KD value of 10⁻⁵ to 10⁻¹²moles/liter or less and binds to related cytokines with a KD valuegreater than 10⁻⁴ moles/liter. Examples of IL13 related targets arehuman IL4. Examples of related cytokines for TSLP are human IL7. Thus,in an embodiment of the present technology, at least two ISVDs comprisedin the polypeptide binds to IL13 with a KD value of 10⁻⁵ to 10⁻¹²moles/liter or less and binds to IL4 of the same species with a KD valuegreater than 10⁻⁴ moles/liter, and at least two ISVDs comprised in thepolypeptide bind to TSLP with a KD value of 10⁻⁵ to 10⁻¹² moles/liter orless and binds to human IL7 of the same species with a KD value greaterthan 10⁻⁴ moles/liter.

Thus, the polypeptide of the present technology has at least half thebinding affinity, or at least the same binding affinity, to human IL13and to human TSLP as compared to a polypeptide consisting of the aminoacid of SEQ ID NO: 1, wherein the binding affinity is measured using thesame method, such as SPR.

Specific binding to a certain target from a certain species does notexclude that the binding unit can also specifically bind to theanalogous target from a different species. For example, specific bindingto human IL13 does not exclude that the binding unit (or a polypeptidecomprising the same) can also specifically bind to IL13 from cynomolgusmonkeys. Likewise, for example, specific binding to human TSLP does notexclude that the binding unit (or a polypeptide comprising the same) canalso specifically bind to TSLP from cynomolgus monkeys (“cyno”).

Specific binding of a binding unit to its designated target can bedetermined in any suitable manner known per se, including, for example,Scatchard analysis and/or competitive binding assays, such asradioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwichcompetition assays, and the different variants thereof known per se inthe art; as well as the other techniques mentioned herein.

The dissociation constant may be the actual or apparent dissociationconstant, as will be clear to the skilled person. Methods fordetermining the dissociation constant will be clear to the skilledperson, and for example include the techniques mentioned below. In thisrespect, it will also be clear that it may not be possible to measuredissociation constants of more than 10⁻⁴ moles/liter or 10⁻³ moles/liter(e.g. of 10⁻² moles/liter). Optionally, as will also be clear to theskilled person, the (actual or apparent) dissociation constant may becalculated on the basis of the (actual or apparent) association constant(KA), by means of the relationship [KD=1/KA].

The affinity of a molecular interaction between two molecules can bemeasured via different techniques known per se, such as the well-knownsurface plasmon resonance (SPR) biosensor technique (see for exampleOber et al. 2001, Intern. Immunology 13: 1551-1559). The term “surfaceplasmon resonance”, as used herein, refers to an optical phenomenon thatallows for the analysis of real-time biospecific interactions bydetection of alterations in protein concentrations within a biosensormatrix, where one molecule is immobilized on the biosensor chip and theother molecule is passed over the immobilized molecule under flowconditions yielding k_(on), k_(off) measurements and hence K_(D) (orK_(A)) values. This can for example be performed using the well-knownBIAcore® system (BIAcore International AB, a GE Healthcare company,Uppsala, Sweden and Piscataway, N.J.). For further descriptions, seeJonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991Biotechniques 11: 620-627), Johnsson et al. (1995, J. Mol. Recognit. 8:125-131), and Johnnson et al. (1991, Anal. Biochem. 198: 268-277).

Another well-known biosensor technique to determine affinities ofbiomolecular interactions is bio-layer interferometry (BLI) (see forexample Abdiche et al. 2008, Anal. Biochem. 377: 209-217). The term“bio-layer Interferometry” or “BLI”, as used herein, refers to alabel-free optical technique that analyzes the interference pattern oflight reflected from two surfaces: an internal reference layer(reference beam) and a layer of immobilized protein on the biosensor tip(signal beam). A change in the number of molecules bound to the tip ofthe biosensor causes a shift in the interference pattern, reported as awavelength shift (nm), the magnitude of which is a direct measure of thenumber of molecules bound to the biosensor tip surface. Since theinteractions can be measured in real-time, association and dissociationrates and affinities can be determined. BLI can for example be performedusing the well-known Octet® Systems (ForteBio, a division of Pall LifeSciences, Menlo Park, USA).

Alternatively, affinities can be measured in Kinetic Exclusion Assay(KinExA) (see for example Drake et al. 2004, Anal. Biochem., 328:35-43), using the KinExA® platform (Sapidyne Instruments Inc, Boise,USA). The term “KinExA”, as used herein, refers to a solution-basedmethod to measure true equilibrium binding affinity and kinetics ofunmodified molecules. Equilibrated solutions of an antibody/antigencomplex are passed over a column with beads precoated with antigen (orantibody), allowing the free antibody (or antigen) to bind to the coatedmolecule. Detection of the antibody (or antigen) thus captured isaccomplished with a fluorescently labeled protein binding the antibody(or antigen).

The GYROLAB® immunoassay system provides a platform for automatedbioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis5: 1765-74).

5.4 (In Vivo) Half-Life Extension

The polypeptide may further comprise one or more other groups, residues,moieties or binding units, optionally linked via one or more peptidiclinkers, in which said one or more other groups, residues, moieties orbinding units provide the polypeptide with increased (in vivo)half-life, compared to the corresponding polypeptide without said one ormore other groups, residues, moieties or binding units. In vivohalf-life extension means, for example, that the polypeptide has anincreased half-life in a mammal, such as a human subject, afteradministration. Half-life can be expressed for example as t½beta.

The type of groups, residues, moieties or binding units is not generallyrestricted and may for example be chosen from the group consisting of apolyethylene glycol molecule, serum proteins or fragments thereof,binding units that can bind to serum proteins, an Fc portion, and smallproteins or peptides that can bind to serum proteins.

More specifically, said one or more other groups, residues, moieties orbinding units that provide the polypeptide with increased half-life canbe chosen from the group consisting of binding units that can bind toserum albumin, such as human serum albumin, or a serum immunoglobulin,such as IgG. In one embodiment, said one or more other binding unitsthat provide the polypeptide with increased half-life is a binding unitthat can bind to human serum albumin. In one embodiment, the bindingunit is an ISVD.

For example, WO 04/041865 describes Nanobodies® binding to serum albumin(and in particular against human serum albumin) that can be linked toother proteins (such as one or more other Nanobodies binding to adesired target) in order to increase the half-life of said protein.

The international application WO 06/122787 describes a number ofNanobodies® against (human) serum albumin. These Nanobodies® include theNanobody® called Alb-1 (SEQ ID NO: 52 in WO 06/122787) and humanizedvariants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 06/122787). Again,these can be used to extend the half-life of therapeutic proteins andpolypeptide and other therapeutic entities or moieties.

Moreover, WO2012/175400 describes a further improved version of Alb-1,called Alb-23.

In one embodiment, the polypeptide comprises a serum albumin bindingmoiety selected from Alb-1, Alb-3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8,Alb-9, Alb-10 and Alb-23. In one embodiment, the serum albumin bindingmoiety is Alb-8 or Alb-23 or its variants, as shown in pages 7-9 ofWO2012/175400 and the albumin binders described in WO 2012/175741,WO2015/173325, WO2017/080850, WO2017/085172, WO2018/104444,WO2018/134235, WO2018/134234. Some serum albumin binders are also shownin Table A-4. In one embodiment, a further component of the polypeptideof the present technology is as described in item E:

E. An ISVD that binds to human serum albumin and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 10 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 10;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 15 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 15; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 20 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 20.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 10, a CDR2 that is the amino acid        sequence of SEQ ID NO: 15 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 20.

Examples of such an ISVD that binds to human serum albumin have one ormore, or all, framework regions as indicated for construct ALB23002 inTable A-2 (in addition to the CDRs as defined in the preceding item E).In one embodiment, it is an ISVD comprising or consisting of the fullamino acid sequence of construct ALB23002 (SEQ ID NO: 5, see Table A-1and A-2).

Item E can be also described using the Kabat definition as:

E′. An ISVD that binds to human serum albumin and comprises

-   -   i. a CDR1 that is the amino acid sequence of SEQ ID NO: 40 or an        amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 40;    -   ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 45 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 45; and    -   iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 20 or        an amino acid sequence with 2 or 1 amino acid difference(s) with        SEQ ID NO: 20.    -   In one embodiment, the ISVD comprises a CDR1 that is the amino        acid sequence of SEQ ID NO: 40, a CDR2 that is the amino acid        sequence of SEQ ID NO: 45 and a CDR3 that is the amino acid        sequence of SEQ ID NO: 20.

Examples of such an ISVD that binds to human serum albumin have one ormore, or all, framework regions as indicated for construct ALB23002 inTable A-2-1 (in addition to the CDRs as defined in the preceding itemE′). In one embodiment, it is an ISVD comprising or consisting of thefull amino acid sequence of construct ALB23002 (SEQ ID NO: 5, see TableA-1 and A-2-1).

In a further embodiment, the amino acid sequence of an ISVD binding tohuman serum albumin may have a sequence identity of more than 90%, suchas more than 95% or more than 99%, with SEQ ID NO: 5, wherein the CDRsare as defined in the preceding item E or E′. In one embodiment, theISVD binding to human serum albumin has the amino acid sequence of SEQID NO: 5.

When such an ISVD binding to human serum albumin has 2 or 1 amino aciddifference in at least one CDR relative to a corresponding reference CDRsequence (item E above), the ISVD has at least half the bindingaffinity, or at least the same binding affinity to human serum albuminas construct ALB23002 set forth in SEQ ID NO: 5, wherein the bindingaffinity is measured using the same method, such as SPR.

In one embodiment, when such an ISVD binding to human serum albumin hasa C-terminal position it exhibits a C-terminal alanine (A) or glycine(G) extension. In one embodiment, such and ISVD is selected from SEQ IDNOs: 59, 60, 62, 64, 65, 66, 67, 68, 69 and 71 (see table A-4 below). Inone embodiment, the ISVD binding to human serum albumin has anotherposition than the C-terminal position (i.e. is not the C-terminal ISVDof the polypeptide of the present technology). In one embodiment, suchan ISVD is selected from SEQ ID NOs: 5, 57, 58, 61, and 63 (see tableA-4 below).

5.5 Nucleic Acid Molecules

Also provided is a nucleic acid molecule encoding the polypeptide of thepresent technology.

A “nucleic acid molecule” (used interchangeably with “nucleic acid”) isa chain of nucleotide monomers linked to each other via a phosphatebackbone to form a nucleotide sequence. A nucleic acid may be used totransform/transfect a host cell or host organism, e.g. for expressionand/or production of a polypeptide. Suitable hosts or host cells forproduction purposes will be clear to the skilled person, and may forexample be any suitable fungal, prokaryotic or eukaryotic cell or cellline or any suitable fungal, prokaryotic or eukaryotic organism. A hostor host cell comprising a nucleic acid encoding the polypeptide of thepresent technology is also encompassed by the present technology.

A nucleic acid may be for example DNA, RNA, or a hybrid thereof, and mayalso comprise (e.g. chemically) modified nucleotides, like PNA. It canbe single- or double-stranded. In one embodiment, it is in the form ofdouble-stranded DNA. For example, the nucleotide sequences of thepresent technology may be genomic DNA, cDNA.

The nucleic acids of the present technology can be prepared or obtainedin a manner known per se, and/or can be isolated from a suitable naturalsource. Nucleotide sequences encoding naturally occurring (poly)peptidescan for example be subjected to site-directed mutagenesis, so as toprovide a nucleic acid molecule encoding polypeptide with sequencevariation. Also, as will be clear to the skilled person, to prepare anucleic acid, also several nucleotide sequences, such as at least onenucleotide sequence encoding a targeting moiety and for example nucleicacids encoding one or more linkers can be linked together in a suitablemanner.

Techniques for generating nucleic acids will be clear to the skilledperson and may for instance include, but are not limited to, automatedDNA synthesis; site-directed mutagenesis; combining two or morenaturally occurring and/or synthetic sequences (or two or more partsthereof), introduction of mutations that lead to the expression of atruncated expression product; introduction of one or more restrictionsites (e.g. to create cassettes and/or regions that may easily bedigested and/or ligated using suitable restriction enzymes), and/or theintroduction of mutations by means of a PCR reaction using one or more“mismatched” primers.

5.6 Vectors

Also provided is a vector comprising the nucleic acid molecule encodingthe polypeptide of the present technology. A vector as used herein is avehicle suitable for carrying genetic material into a cell. A vectorincludes naked nucleic acids, such as plasmids or mRNAs, or nucleicacids embedded into a bigger structure, such as liposomes or viralvectors.

Vectors generally comprise at least one nucleic acid that is optionallylinked to one or more regulatory elements, such as for example one ormore suitable promoter(s), enhancer(s), terminator(s), etc.). In oneembodiment, the vector is an expression vector, i.e. a vector suitablefor expressing an encoded polypeptide or construct under suitableconditions, e.g. when the vector is introduced into a (e.g. human) cell.For DNA-based vectors, this usually includes the presence of elementsfor transcription (e.g. a promoter and a polyA signal) and translation(e.g. Kozak sequence).

In one embodiment, in the vector, said at least one nucleic acid andsaid regulatory elements are “operably linked” to each other, by whichis generally meant that they are in a functional relationship with eachother. For instance, a promoter is considered “operably linked” to acoding sequence if said promoter is able to initiate or otherwisecontrol/regulate the transcription and/or the expression of a codingsequence (in which said coding sequence should be understood as being“under the control of” said promotor). Generally, when two nucleotidesequences are operably linked, they will be in the same orientation andusually also in the same reading frame. They will usually also beessentially contiguous, although this may also not be required.

In one embodiment, any regulatory elements of the vector are such thatthey are capable of providing their intended biological function in theintended host cell or host organism.

For instance, a promoter, enhancer or terminator should be “operable” inthe intended host cell or host organism, by which is meant that forexample said promoter should be capable of initiating or otherwisecontrolling/regulating the transcription and/or the expression of anucleotide sequence—e.g. a coding sequence—to which it is operablylinked.

5.7 Compositions

The present technology also provides a composition comprising at leastone polypeptide of the present technology, at least one nucleic acidmolecule encoding a polypeptide of the present technology or at leastone vector comprising such a nucleic acid molecule. The composition maybe a pharmaceutical composition. The composition may further comprise atleast one pharmaceutically acceptable carrier, diluent or excipientand/or adjuvant, and optionally comprise one or more furtherpharmaceutically active polypeptides and/or compounds.

5.8 Host Organisms

The present technology also pertains to host cells or host organismscomprising the polypeptide of the present technology, the nucleic acidencoding the polypeptide of the present technology, and/or the vectorcomprising the nucleic acid molecule encoding the polypeptide of thepresent technology.

Suitable host cells or host organisms are clear to the skilled person,and are for example any suitable fungal, prokaryotic or eukaryotic cellor cell line or any suitable fungal, prokaryotic or eukaryotic organism.Specific examples include HEK293 cells, CHO cells, Escherichia coli orPichia pastoris. In one embodiment, the host is Pichia pastoris.

5.9 Methods and Uses of the Polypeptide

The present technology also provides a method for producing thepolypeptide of the present technology. The method may comprisetransforming/transfecting a host cell or host organism with a nucleicacid encoding the polypeptide, expressing the polypeptide in the host,optionally followed by one or more isolation and/or purification steps.Specifically, the method may comprise:

a) expressing, in a suitable host cell or host organism or in anothersuitable expression system, a nucleic acid sequence encoding thepolypeptide; optionally followed by:

b) isolating and/or purifying the polypeptide.

Suitable host cells or host organisms for production purposes will beclear to the skilled person, and may for example be any suitable fungal,prokaryotic or eukaryotic cell or cell line or any suitable fungal,prokaryotic or eukaryotic organism. Specific examples include HEK293cells, CHO cells, Escherichia coli or Pichia pastoris. In oneembodiment, the host is Pichia pastoris.

The polypeptide of the present technology, a nucleic acid molecule orvector as described, or a composition comprising the polypeptide of thepresent technology, nucleic acid molecule or vector are useful as amedicament.

Accordingly, the present technology provides the polypeptide of thepresent technology, a nucleic acid molecule or vector as described, or acomposition comprising the polypeptide of the present technology,nucleic acid molecule or vector for use as a medicament.

Also provided is the polypeptide of the present technology, a nucleicacid molecule or vector as described, or a composition comprising thepolypeptide of the present technology, nucleic acid molecule or vectorfor use in the (prophylactic or therapeutic). treatment of aninflammatory disease.

Further provided is a (prophylactic and/or therapeutic) method oftreating an inflammatory disease, wherein said method comprisesadministering, to a subject in need thereof, a pharmaceutically activeamount of the polypeptide of the present technology, a nucleic acidmolecule or vector as described, or a composition comprising thepolypeptide of the present technology, nucleic acid molecule or vector.

Further provided is the use of the polypeptide of the presenttechnology, a nucleic acid molecule or vector as described, or acomposition comprising the polypeptide of the present technology,nucleic acid molecule or vector in the preparation of a pharmaceuticalcomposition. In one embodiment, the prepared pharmaceutical compositionis for treating an inflammatory disease.

The inflammatory disease is a type 2 inflammatory disease such as atopicdermatitis and asthma.

A “subject” as referred to in the context of the present technology canbe any animal, and more specifically a mammal. Among mammals, adistinction can be made between humans and non-human mammals. Non-humananimals may be for example companion animals (e.g. dogs, cats),livestock (e.g. bovine, equine, ovine, caprine, or porcine animals), oranimals used generally for research purposes and/or for producingantibodies (e.g. mice, rats, rabbits, cats, dogs, goats, sheep, horses,pigs, non-human primates, such as cynomolgus monkeys, or camelids, suchas llama or alpaca).

In the context of prophylactic and/or therapeutic purposes, the subjectcan be any animal, and more specifically any mammal. In one embodiment,the subject is a human subject.

Substances (including polypeptides, nucleic acid molecules and vectors)or compositions may be administered to a subject by any suitable routeof administration, for example by enteral (such as oral or rectal) orparenteral (such as epicutaneous, sublingual, buccal, nasal,intra-articular, intradermal, intramuscular, intraperitoneal,intravenous, subcutaneous, transdermal, or transmucosal) administration.In one embodiment, substances are administered by parenteraladministration, such as intramuscular, subcutaneous or intradermaladministration. In one embodiment, subcutaneous administration is used.

An effective amount of a polypeptide, a nucleic acid molecule or vectoras described, or a composition comprising the polypeptide, nucleic acidmolecule or vector can be administered to a subject in order to providethe intended treatment results.

One or more doses can be administered. If more than one dose isadministered, the doses can be administered in suitable intervals inorder to maximize the effect of the polypeptide, composition, nucleicacid molecule or vector.

TABLE A-1 Amino acid sequences of the different monovalent V_(HH) building blocks identified within the pentavalent polypeptide F027400161 (“ID” refers to the SEQ ID NO as used herein) Name IDAmino acid sequence 4B02 2 DVQLVESGGGVVQPGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAALSGDGYSTYTANSVKGRFTI SRDNSKNTVYLQMNSLRPEDTALYYCAAKLQYVSGWSYDYPYWGQGTLVTVSS 4B06 3 EVQLVESGGGVVQPGGSLRLSCAASGFTFNNYAMKWVRQAPGKGLEWVSSITTGGGSTDYADSVKGRFTI SRDNSKNTLYLQMNSLRPEDTALYYCANVPFGYYSEHFSGLSFDYRGQGTLVTVSS 501A02 4 EVQLVESGGGVVQPGGSLRLSCAASGSGFGVNILYWYRQAAGIERELIASITSGGITNYVDSVKGRFTIS RDNSENTMYLQMNSLRAEDTGLYYCASRNIFDGTTEWGQGTLVTVSS ALB23002 5 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRFTI SRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS 529F10 6 EVQLVESGGGVVQPGGSLRLSCAASGFTFADYDYDIGWFRQAPGKEREGVSCISNRDGSTYYADSVKGRF TISRDNSKNTVYLQMNSLRPEDTALYYCAVEIHCDDYGVENFDFDPWGQGTLVTVSS

TABLE A-2Sequences for CDRs according to AbM definition and frameworks (“ID” refers to the given SEQ ID NO)ID V_(HH) ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 2 4B02 22DVQLV  7 GRTFS 24 WFRQA 12 ALSGD 29 TANSVKGRFTISR 17 KLQYV 34 WGQGTESGGG SYRMG PGKER GYSTY DNSKNTVYLQMNS SGWSY LVTVS VVQPG EFVALRPEDTALYYCAA DYPY S GSLRL SCAAS 3 4B06 23 EVQLV  8 GFTFN 25 WVRQA 13SITTG 30 YADSVKGRFTISR 18 VPFGY 35 RGQGT ESGGG NYAMK PGKGL GGSTDDNSKNTLYLQMNS YSEHF LVTVS VVQPG EWVS LRPEDTALYYCAN SGLSF S GSLRL DYSCAAS 4 501A02 23 EVQLV  9 GSGFG 26 WYRQA 14 SITSG 31 YVDSVKGRFTISR 19RNIFD 34 WGQGT ESGGG VNILY AGIER GITN DNSENTMYLQMNS GTTE LVTVS VVQPGELIA LRAEDTGLYYCAS S GSLRL SCAAS 5 ALB23002 23 EVQLV 10 GFTFR 27 WVRQA15 SISGS 32 YADSVKGRFTISR 20 GGSLS 36 SSQGT ESGGG SFGMS PGKGP GSDTLDNSKNTLYLQMNS R LVTVS VVQPG EWVS LRPEDTALYYCTI S GSLRL SCAAS 6 529F10 23EVQLV 11 GFTFA 28 WFRQA 16 CISNR 33 YADSVKGRFTISR 21 EIHCD 34 WGQGTESGGG DYDYD PGKER DGSTY DNSKNTVYLQMNS DYGVE LVTVS VVQPG IG EGVSLRPEDTALYYCAV NFDFD S GSLRL P SCAAS

TABLE A-2.1Sequences for CDRs according to Kabat definition and frameworks (“ID” refers to the given SEQ ID NO)ID V_(HH) ID FR1 ID CDR1 ID FR2 ID CDR2 ID FR3 ID CDR3 ID FR4 2 4B02 47DVQLV 37 SYRMG 24 WFRQA 42 ALSGD 52 RFTISRDNSKNTV 17 KLQYV 34 WGQGTESGGG PGKER GYSTY YLQMNSLRPEDTA SGWSY LVTVS VVQPG EFVA TANSV LYYCAA DYPYS GSLRL KG SCAAS GRTFS 3 4B06 48 EVQLV 38 NYAMK 25 WVRQA 43 SITTG 53RFTISRDNSKNTL 18 VPFGY 35 RGQGT ESGGG PGKGL GGSTD YLQMNSLRPEDTA YSEHFLVTVS VVQPG EWVS YADSV LYYCAN SGLSF S GSLRL KG DY SCAAS GFTFN 4 501A0249 EVQLV 39 VNILY 26 WYRQA 44 SITSG 54 RFTISRDNSENTM 19 RNIFD 34 WGQGTESGGG AGIER GITNY YLQMNSLRAEDTG GTTE LVTVS VVQPG ELIA VDSVK LYYCAS SGSLRL G SCAAS GSGFG 5 ALB23002 50 EVQLV 40 SFGMS 27 WVRQA 45 SISGS 55RFTISRDNSKNTL 20 GGSLS 36 SSQGT ESGGG PGKGP GSDTL YLQMNSLRPEDTA R LVTVSVVQPG EWVS YADSV LYYCTI S GSLRL KG SCAAS GFTFR 6 529F10 51 EVQLV 41DYDYD 28 WFRQA 46 CISNR 56 RFTISRDNSKNTV 21 EIHCD 34 WGQGT ESGGG IGPGKER DGSTY YLQMNSLRPEDTA DYGVE LVTVS VVQPG EGVS YADSV LYYCAV NFDFD SGSLRL KG P SCAAS GFTFA

TABLE A-3 Amino acid sequences of selected multivalentpolypeptide (“ID” refers to the given SEQ ID NO) Name IDAmino acid sequence F027400161 1 DVQLVESGGGVVQPGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAALSGDGYSTYTANSVKGRF TISRDNSKNTVYLQMNSLRPEDTALYYCAAKLQYVSGWSYDYPYWGQGTLVTVSSGGGGSGGGGSGGG GSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFNNYAMKWVRQAPGKGLE WVSSITTGGGSTDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCANVPFGYYSEHFSGLSFD YRGQGTLVTVSSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGSGFGVNILYWYRQAAGIEREL IASITSGGITNYVDSVKGRFTISRDNSENTMYLQMNSLRAEDTGLYYCASRNIFDGTTEWGQGTLVTV SSGGGGSGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSD TLYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSE VQLVESGGGVVQPGGSLRLSCAASGFTFADYDYDIGWFRQAPGKEREGVSCISNRDGSTYYADSVKGR FTISRDNSKNTVYLQMNSLRPEDTALYYCAVEIHCDDYGVENFDFDPWGQGTLVTVSSA

TABLE A-4 Serum albumin binding ISVD sequences(“ID” refers to the SEQ ID NO as used herein) Name IDAmino acid sequence Alb8 57 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb23 58 EVQLLESGGGLVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRF TISRDNSKNTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb129 59 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb132 60 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRF TISRDNSKNTLYLQMNSLRPEDTATYYCTIGGSLSRSSQGTLVTVSSA Alb11 61 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSS Alb11 62 EVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGM (S110K)-ASWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVKVSSA Alb82 63 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb82-A 64 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA Alb82-AA 65 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAA Alb82-AAA 66 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSAAA Alb82-G 67 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSG Alb82-GG 68 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGG Alb82-GGG 69 EVQLVESGGGVVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRF TISRDNAKTTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSGGG Alb23002  5 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRF TISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSS Alb223 71 EVQLVESGGGVVQPGGSLRLSCAASGFTFRSFGMSWVRQAPGKGPEWVSSISGSGSDTLYADSVKGRF TISRDNSKNTLYLQMNSLRPEDTALYYCTIGGSLSRSSQGTLVTVSSA

TABLE A-5 Linker sequences (“ID” refers to the SEQ ID NO as used herein)Name ID Amino acid sequence   3A linker 72 AAA  5GS linker 73 GGGGS 7GS linker 74 SGGSGGS  8GS linker 75 GGGGSGGS  9GS linker 76 GGGGSGGGS10GS linker 77 GGGGSGGGGS 15GS linker 78 GGGGSGGGGSGGGGS 18GS linker 79GGGGSGGGGSGGGGSGGS 20GS linker 80 GGGGSGGGGSGGGGSGGGGS 25GS linker 81GGGGSGGGGSGGGGSGGGGSGGGGS 30GS linker 82 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS35GS linker 83 GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGS 40GS linker 84GGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGS G1 hinge 85 EPKSCDKTHTCPPCP9GS-G1 hinge 86 GGGGSGGGSEPKSCDKTHTCPPCP Llama upper  87 EPKTPKPQPAAAlong hinge  region G3 hinge 88 ELKTPLGDTTHTCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCPEPKSCDTPPPCPRCP

TABLE A-6 Amino acid sequences of selected monospecific multivalent polypeptides(“ID” refers to the given SEQ ID NO) Name ID Amino acid sequenceF010700003 148 EVQLVESGGGLVQPGGSLRLSCAASGFTFN [F0107004606-NYAMKWVRQAPGKGLEWVSSITTGGGSTDY 35GS- ADSVKGRFTISRDNRKNTLYLQMNSLKPEDF0107004B02]¹ TAVYYCANVPFGYYSEHFSGLSFDYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVA ALSGDGYSTYTANSVNSRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAKLQYVSGWSYD YPYWGQGTLVTVSS F010700014 149EVQLVESGGGLVQAGGSLRLSCAASGRTFS [F0107004B02-SYRMGWFRQAPGKEREFVAALSGDGYSTYT 35GS- ANSVNSRFTISRDNAKNTVYLQMNSLKPEDF0107004B02]¹ TAIYYCAAKLQYVSGWSYDYPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSEVQLVESGGGLVQAGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAALS GDGYSTYTANSVNSRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAKLQYVSGWSYDYPY WGQGTLVTVSS F010700029 150EVQLVESGGGLVQAGGSLRLSCAASGRTFS [F0107004B02-SYRMGWFRQAPGKEREFVAALSGDGYSTYT 35GS- ANSVNSRFTISRDNAKNTVYLQMNSLKPEDF0107004B06]¹ TAIYYCAAKLQYVSGWSYDYPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMKWVRQAPGKGLEWVSSIT TGGGSTDYADSVKGRFTISRDNRKNTLYLQMNSLKPEDTAVYYCANVPFGYYSEHFSGLS FDYRGQGTLVTVSS F010700031 151EVQLVESGGGLVQPGGSLRLSCAASGFTFN [F0107004B06-NYAMKWVRQAPGKGLEWVSSITRTGGGSTD 35GS- YADSVKGRFTISRDNKNTLYLQMNSLKPEDF0107004B06]¹ TAVYYCANVPFGYYSEHFSGLSFDYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMKWVRQAPGKGLEWVS SITTGGGSTDYADSVKGRFTISRDNRKNTLYLQMNSLKPEDTAVYYCANVPFGYYSEHFS GLSFDYRGQGTLVTVSS F010703842 152EVQLVESGGGLVQAGGSLRLSCAASGFTFA [F0107529F10-DYDYDIGWFRQAPGKEREGVSCISNRDGST 35GS- YYTDSVKGRFTISSDNAKNTVSLQMNSLKPF0107501A02]¹ EDTAVYYCAVEIHCDDYGVENFDFDPWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGG GSGGGGSGGGGSEVQLVESGGGLVQAGESLRLSCAASGSGFGVNILYWYRQAAGIERELI ASITSGGITNYVDSVKGRFTISRDNAENTMYLQMNSLKAEDTGVYYCASRNIFDGTTEWG QGTLVTVSS F027400016 153DVQLVESGGGVVQPGGSLRLSCAASGSGFG [F0107501A02VNILYWYRQAPGKQRELIASITSGGITNYV (E1D, L11V, DSVKGRFTISRDNSKNTLYLQMNSLRPEDT A14P, E16G, ALYYCASRNIFDGTTEWGQGTLVTVSSGGG A41P, I43K, GSGGGGSGGGGSGGGGSGGGGSGGGGSGGG E44Q, A74S, GSEVQLVESGGGVVQPGGSLRLSCAASGFT E75K, M78L, FADYDYDIGWFRQAPGKEREGVSCISNRDG K83R, A84P, STYYADSVKGRFTISRDNSKNTVYLQMNSL G88A,  RPEDTALYYCAVEIHCDDYGVENFDFDPWGV89L)*-35GS- QGTLVTVSS 529F10)]¹ F010700003- 154EVQLVESGGGVVQPGGSLRLSCAASGFTFN SO[4B06-35GS-NYAMKWVRQAPGKGLEWVSSITTGGGSTDY 4B02(D1E)*]¹ADSVKGRFTISRDNSKNTLYLQMNSLRPED TALYYCANVPFGYYSEHFSGLSFDYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVA ALSGDGYSTYTANSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAKLQYVSGWSYD YPYWGQGTLVTVSS F010700014- 155DVQLVESGGGVVQPGGSLRLSCAASGRTFS SO[4B02-35GS-SYRMGWFRQAPGKEREFVAALSGDGYSTYT 4B02(D1E)*]¹ANSVKGRFTISRDNSKNTVYLQMNSLRPED TALYYCAAKLQYVSGWSYDYPYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGG GGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGRTFSSYRMGWFRQAPGKEREEVAALS GDGYSTYTANSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAKLQYVSGWSYDYPY WGQGTLVTVSS F010700029- 156DVQLVESGGGVVQPGGSLRLSCAASGRTFS SO[4B02-35GS-SYRMGWFRQAPGKEREFVAALSGDGYSTYT 4B06]¹ ANSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAAKLQYVSGWSYDYPYWGQGTLVT VSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSC AASGFTFNNYAMKWVRQAPGKGLEWVSSITTGGGSTDYADSVKGRFTISRDNSKNTLYLQ MNSLRPEDTALYYCANVPFGYYSEHFSGLSFDYRGQGTLVTVSS F010700031- 157 EVQLVESGGGVVQPGGSLRLSCAASGFTFNSO[4B06-35GS- NYAMKWVRQAPGKGLEWVSSITTGGGSTDY 4B06]¹ADSVKGRFTISRDNSKNTLYLQMNSLRPED TALYYCANVPFGYYSEHFSGLSFDYRGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGG SGGGGSGGGGSEVQLVESGGGVVQPGGSLRLSCAASGFTFNNYAMKWVRQAPGKGLEWVS SITTGGGSTDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCANVPFGYYSEHFS GLSFDYRGQGTLVTVSS F010703842- 158EVQLVESGGGVVQPGGSLRLSCAASGFTFA SO[529F10- DYDYDIGWFRQAPGKEREGVSCISNRDGST35GS-501A02]¹ YYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAVEIHCDDYGVENFDFDPWGQG TLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGVVQPGGSL RLSCAASGSGFGVNILYWYRQAAGIERELIASITSGGITNYVDSVKGRFTISRDNSENTM YLQMNSLRAEDTGLYYCASRNIFDGTTEWG QGTLVTVSSF027400016- 159 EVQLVESGGGVVQPGGSLRLSCAASGSGFG SO[501A02-VNILYWYRQAAGIERELIASITSGGITNYV 35GS-529F10]¹DSVKGRFTISRDNSENTMYLQMNSLRAEDT GLYYCASRNIFDGTTEWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGG GSEVQLVESGGGVVQPGGSLRLSCAASGFTFADYDYDIGWFRQAPGKEREGVSCISNRDG STYYADSVKGRFTISRDNSKNTVYLQMNSLRPEDTALYYCAVEIHCDDYGVENFDFDPWG QGTLVTVSS ¹[ ] indicates the monovalentbuilding blocks and their linkage which were used for the bivalentconstruct. *( ) indicates the amino acid substitutions introduced intothe (parental) monovalent building block (e.g.: 4B02(D1E) means that themonovalent building block 4B02 (SEQ ID NO: 2) contains a D1Esubstitution).

6. EXAMPLES

6.1 Generation of Monovalent ISVDs Specifically Binding to IL-13 andTSLP, Respectively

6.1.1 Example 1: Immunizations

Three llamas were immunized with recombinant human IL-13 (Peprotech, catnr 200-13, E. coli-derived) according to standard protocols, with theaim to induce a heavy-chain antibody dependent humoral immune response.In addition, these llamas were boosted with human/cyno IL-13 fromanother source (Sino Biological, cat nrs 10369-HNAC and 11057-CNAH,mammalian cell derived).

Another three llamas were immunized with recombinant hTSLP-Fc. Inaddition, these llamas were boosted with cyno TSLP-Fc. Two additionalllamas were immunized with hTSLP alternated with cyno TSLP-Fc.

Immune blood (PBL) samples were taken at regular intervals, and totalRNA was prepared from the isolated B-cells. The humoral immune responsewas monitored during the immunization process by comparing the antigenspecific serum titers of a sample collected prior to initiation ofimmunization and a serum sample typically collected after multipleantigen administrations. Briefly, 96-well Maxisorp plates were coatedwith human IL-13 (Sino Biological, cat nr 10369-HNAC) or human TSLP.Recombinant human TSLP is commercially available, such as from R&DSystems (cat nr 1398-TS). After blocking and adding diluted serumsamples, the presence of anti-IL-13 and anti-TSLP ISVDs was demonstratedby using HRP (horseradish peroxidase) conjugated goat anti-llamaimmunoglobulin (Bethyl Laboratories Inc.) and a subsequent enzymaticreaction in the presence of the substrate TMB(3,3′,5,5′-tetramentylbenzidine).

6.1.2 Example 2: Library Construction and Phage Display Selections

Peripheral blood mononuclear cells were prepared from the blood samplesusing Ficoll-Hypaque according to the manufacturer's instructions. TotalRNA extracted from these cells and from lymph nodes was used as startingmaterial for RT-PCR to amplify ISVD encoding gene fragments. Thesefragments were cloned into phagemid vector pAX212. Phage was preparedaccording to standard protocols (Antibody Phage Display: Methods andProtocols (First Edition, 2002, O'Brian and Aitken eds., Humana Press,Totowa, N.J.) and stored after filter sterilization at 4° C. untilfurther use. Five phage libraries were constructed for IL13, withlibrary sizes between 6.1×10⁸ and 1.3×10⁹, and a percentage of insertranging from 87 to 96%. Five phage libraries were constructed for TSLP,with library sizes between 4.2×10⁸ and 9.8×10⁸, and a percentage ofinsert ranging from 91 to 100%.

To identify ISVDs recognizing human and cyno IL-13, the phage librarieswere incubated with 50 nM soluble biotinylated hIL13-Fc in presence ofIgG from human serum (Sigma, 14506). Complexes of hIL-13-Fc and phagewere captured from solution on streptavidin coated magnetic beads. Afterextensive washing with PBS/0.05% Tween20, bound phage were eluted byaddition of trypsin (1 mg/ml). Outputs of these round 1 selections wereincubated with 0.05, 0.5 or 5 nM soluble biotinylated hIL-13-Fc or cynoIL-13-Fc. Not enriched outputs from the round 2 selections were furtherincubated with a 0.005, 0.05, 0.5 or 5 nM soluble biotinylated human orcyno IL-13-Fc in round 3. Individual clones from enriched round 2 andround 3 selections were picked.

To identify ISVDs recognizing human and cyno TSLP, the phage librarieswere incubated with 50 nM soluble biotinylated hTSLP-Fc in presence ofIgG from human serum (Sigma, 14506) or with 500 nM biotinylated hTSLP orwith 500 nM biotinylated cyno TSLP. The human and cyno TSLP sequencesare known (Uniprot accession Uniprot accession Q969D9 and NCBI RefSeqXP_005557555.1, respectively). Recombinant protein was used to performthe assay. Complexes of TSLP and phage were captured from solution onstreptavidin coated magnetic beads. After extensive washing withPBS/0.05% Tween20, bound phage were eluted by addition of trypsin (1mg/ml). Outputs of these round 1 selections were incubated with 5 nMsoluble biotinylated hILTSLP-Fc or cyno TLSP-Fc or 0.5 nM biotinylatedhTSLP. Outputs from the round 2 selections were incubated with 0.05, 0.5or 5 nM soluble biotinylated human TSLP, TSLP-Fc or cyno TSLP-Fc. Fromeach round individual clones from the enriched outputs were picked.

All individual clones were grown in 96 deep well plates (1 ml volume).ISVD expression was induced by adding IPTG to a final concentration of 1mM. Periplasmic extracts were prepared by freezing the cell pellets anddissolving them in 100 μPBS. Cell debris was removed by centrifugation.

As a control, selected periplasmic extracts were screened in an ELISAfor binding to human and cyno IL13-Fc, respectively TSLP-Fc. Theassessment was performed in a Spectraplate 384-HB (PerkinElmer) in a 25μl format. The antigens were coated overnight at 1 μg/ml in PBS at 4° C.Wells were blocked with a casein solution (1%). After addition of a5-fold dilution of peri plasmic extracts, ISVD binding was detectedusing mouse anti-Flag-HRP (Sigma) and a subsequent enzymatic reaction inthe presence of substrate esTMB (3,3′,5,5′-tetramentylbenzidine).

6.1.3 Example 3: Screening for Blocking ISVDs in Periplasmic Extracts byAlphaScreen Assays Using Human IL13 and Human TSLP

In order to determine the blocking capacity of the ISVDs, crudeperiplasmic extracts were screened in different protein-basedcompetition assays using the AlphaScreen technology (PerkinElmer,Waltham, Mass. USA). Fluorescence was measured using the EnVisionMultilabel Plate Reader (PerkinElmer) using an excitation wavelength of680 nm and an emission wavelength of 520 nm.

In the hIL-13:hIL-13Rα1 binary complex AlphaScreen, it was investigatedif ISVDs could block the interaction between hIL-13 and the hIL-13Rα1extracellular domain. To this end, dilutions of the periplasmic extractswere pre-incubated with biotinylated hIL-13 (Peprotech cat nr 200-13).To this mixture, hIL-13Rα1-hFc (R&D systems; cat nr 146-IR) and antihFc-coupled Acceptor beads were added and further incubated for 1 hourat room temperature, followed by addition of streptavidin-coupled Donorbeads and an additional 1-hour incubation. When the binary complex isformed, Acceptor and Donor beads are brought into proximity and uponlaser excitation a detectable signal is generated. Decrease in theAlphaScreen signal indicates that the binding of biotinylated hIL-13 tohIL-13Rα1 is blocked by the ISVD present in the periplasmic extract. Ina similar set-up it was investigated if ISVDs could block theinteraction between hTSLP (eBioscience, cat nr 10-8499) and the hTSLPRextracellular domain (R&D systems cat nr 981-TR).

In the hIL-13:hIL-13Rα1:hIL4Rα ternary complex AlphaScreen, it wasscreened if ISVDs could block the recruitment of hIL4Rα to thehIL13:hIL13Rα1 binary complex. hIL-13 binds to hIL-13Rα1 and this binarycomplex recruits hIL4Rα, resulting in formation of the ternary complexhIL-13:hIL-13Rα1:hIL4Rα. Dilutions of the periplasmic extracts werepre-incubated with hIL-13 (Peprotech cat nr 200-13) and biotinylatedhuIL4Rα (R&D Systems; cat nr 230-4R/CF). To this mixture, hIL-13Rα1-hFc(R&D systems; cat nr 146-IR) and anti hFc-coupled Acceptor beads wereadded and further incubated for 1 hour at room temperature, followed byaddition of streptavidin-coupled Donor beads and an additional 1-hourincubation. When the ternary complex is formed, Acceptor and Donor beadsare brought into proximity and upon laser excitation a detectable signalis generated. Decrease in the AlphaScreen signal indicates that theformation of the ternary complex is blocked by the ISVD present in theperiplasmic extract. Similarly, it was investigated if ISVDs could blockthe formation of the hTSLP:hTSLPR:hIL7Rα complex (hIL7Rα source=SinoBiological cat nr 1095-H08H).

Based on the Alphascreen analysis (Table 3 and Table 4), a number ofblocking ISVDs were selected and sequenced (Table 1 and Table 2).

TABLE 1 Amino acid sequences of  functional anti-IL-13 ISVDs. ISVD IDSEQUENCE F0107004B02 EVQLVESGGGLVQAGGSLRLSCAASGRTFSSYRMGWF (SEQ ID RQAPGKEREFVAALSGDGYSTYTANSVNSRFTISRDN NO: 144)AKNTVYLQMNSLKPEDTAIYYCAAKLQYVSGWSYDYP YWGQGTLVTVSS F0107004B06EVQLVESGGGLVQPGGSLRLSCAASGFTFNNYAMKWV (SEQ ID RQAPGKGLEWVSSITTGGGSTDYADSVKGRFTISRDN NO: 145)RKNTLYLQMNSLKPEDTAVYYCANVPFGYYSEHFSGL SFDYRGQGTLVTVSS

TABLE 2 Amino acid sequences  of functional anti-TSLP ISVDs. ISVD IDSEQUENCE F0107501A02 EVQLVESGGGLVQAGESLRLSCAASGSGFGVNILYWY (SEQ ID RQAAGIERELIASITSGGITNYVDSVKGRFTISRDNA NO: 146)ENTMYLQMNSLKAEDTGVYYCASRNIFDGTTEWGQGT LVTVSS F0107529F10EVQLVESGGGLVQAGGSLRLSCAASGFTFADYDYDIG (SEQ ID WFRQAPGKEREGVSCISNRDGSTYYTDSVKGRFTISS NO: 147)DNAKNTVSLQMNSLKPEDTAVYYCAVEIHCDDYGVEN FDFDPWGQGTLVTVSS

6.1.4 Example 4: Surface Plasmon Resonance Analysis of PeriplasmicExtracts on IL13 and TSLP

Off-rates of the periplasmic extracts containing anti-IL13 or anti-TSLPISVDs were measured by Surface Plasmon Resonance (SPR) using a ProteonXPR36 instrument (Bio-Rad Laboratories, Inc.). Phosphate buffered saline(PBS), pH7.4 supplemented with 0.005% Tween20 was used as running bufferand the experiments were performed at 25° C.

hIL13-Fc, cyno IL13-Fc, hTSLP-Fc and cyno TSLP-Fc were immobilized onProteOn GLC Sensor Chips by amine coupling using EDC and NHS at flowrate 30 μl/min for activation. IL13 proteins were injected at 10 μg/mlin ProteOn Acetate buffer at pH5.0. TSLP proteins were injected at 5μg/mL in ProteOn Acetate Buffer at pH5.5. After immobilization, surfaceswere deactivated with ethanolamine.

Periplasmic extracts of the ISVD candidates were diluted 10 times inPBS-Tween20 (0.1%) and injected for 2 minutes at 45 μl/min and allowedto dissociate for 900 seconds. Between different samples, the surfaceswere regenerated with a 2 minute injection of Phosphoric Acid (0.425%)at 45 μl/min, in case of IL13, or with a 1 minute injection of GlycinepH3.0 (5 mM)/SDS(0.25%) at 45 μL/min, in case of TSLP. From thesensorgrams obtained for the different periplasmic extracts off-rateswere calculated.

Off-rate analysis on hIL-13-Fc and cyno IL-13-Fc is shown in Table 3.

TABLE 3 Summary of the screening results of anti-IL-13 ISVDs F0107004B02and F0107004B06. IL-13-IL-13Rα1 IL13-IL-13Rα1-IL4Rα AlphaScreenAlphaScreen k_(off) hIL-13-Fc k_(off) cyno IL-13-Fc ISVD ID (%inhibition) (% inhibition) (1/s) (1/s) F0107004B02 37 61 1.7E−03 1.1E−03F0107004B06 21 66 6.3E−03 8.9E−03

Off-rate analysis on hTSLP-Fc and cyno TSLP-Fc is shown in Table 4.

TABLE 4 Summary of the screening results of anti-TSLP ISVDs F0107501A02and F0107529F10. TSLP-TSLPR TSLP-TSLPR-IL7Rα AlphaScreen AlphaScreenk_(off) hTSLP-Fc k_(off) cyno TSLP-Fc ISVD ID (% inhibition) (%inhibition) (1/s) (1/s) F0107501A02 92 97 3.6E−05 6.6E−04 F0107529F10 9999  <5E−05 1.8E−03

6.1.5 Example 5: Expression and Purification of Anti-IL-13 and Anti-TSLPISVDs

Anti-IL-13 ISVDs and anti-TSLP ISVDs were selected for expression andpurification, based on their blocking capacity in AlphaScreen assays andoff-rate values. Sequences are shown in Tables 1 and 2.

ISVDs were expressed in E. coli TG1 cells as c-myc, His6-taggedproteins. Expression was induced by addition of 1 mM IPTG and allowed tocontinue for 3h at 37° C. After spinning the cell cultures, periplasmicextracts were prepared by freeze-thawing the pellets and resuspension indPBS. These extracts were used as starting material for immobilizedmetal affinity chromatography (IMAC) using Nickel Sepharose™ 6 FFcolumns (Atoll). ISVDs were eluted from the column with 250 mM imidazoleand subsequently desalted towards dPBS. For the cell-based assaysdescribed below, endotoxins were removed by gel filtration in thepresence of 50 mM Octylβ-D-glucopyranoside (OGP, Sigma). Endotoxinlevels were determined using a standard LAL-assay.

6.1.6 Example 6: Blocking Capacity of Purified Anti-IL13 and Anti-TSLPISVDs in AlphaScreen Assays

The binary and ternary complex AlphaScreen assays, as described inexample 3, were used to determine the IC50 values of the anti-IL-13 andanti-TSLP ISVDs, purified as described in Example 5. Instead ofdilutions of the periplasmic extracts, a dilution series of eachpurified ISVD starting from 500 nM down to 1.8 pM was pre-incubated withIL-13 or TSLP.

The tested anti-IL-13 ISVDs inhibited the formation of the ternarycomplex and partially blocked the binary complex with IC50 values asshown in Table 5.

TABLE 5 IC50 values for the blocking anti-IL13 ISVDs as determined inthe binary and ternary complex AlphaScreen assays. IL-13-IL-13Rα1AlphaScreen IL-13-IL-13Rα1-IL4Rα AlphaScreen ISVD ID IC50 (M) %Inhibition IC50 (M) % Inhibition F0107004B02 4.0E−09 82 1.7E−09 100F0107004B06 6.1E−08 55 6.7E−09 100

The tested anti-TSLP ISVDs fully inhibited the formation of the ternarycomplex, and also inhibited the formation of the binary complex, withIC50 values as shown in Table 6.

TABLE 6 IC50 values for the blocking anti-TSLP ISVDs as determined inthe binary and ternary complex AlphaScreen assays. TSLP-TSLPRAlphaScreen TSLP-TSLPR-IL7Rα AlphaScreen ISVD ID IC50 (M) % InhibitionIC50 (M) % Inhibition F0107501A02 3.00E−10 97 3.50E−10 100 F0107529F101.30E−11 100 1.80E−10 100

6.1.7 Example 7: Blocking Capacity of Purified Anti-IL-13 and Anti-TSLPISVDs in Cell-Based Assays

The inhibitory potency of the anti-IL-13 ISVDs was determined in acell-based assay monitoring IL-13 mediated proliferation of TF-1 cells.To this end, TF-1 cells were cultured in RPMI 1640 medium with theaddition of ⅕ HEPES, 1/500 Na-Pyruvate, 1/500 Glutamax and 2 ng/mLrecombinant human GM-CSF. TF-1 cells were seeded at 40.000 cells perwell in growth medium w/o GM-CSF. A dilution series of the purifiedanti-IL-13 ISVDs or reference compounds was added. After 15 minincubation at 37° C., 200 pM of IL-13 (Peprotech cat nr 200-13) wasadded. After 96 hours, proliferation of the TF-1 cells was determinedwith Cell Titer 96 Aqueous One Solution (Promega #G3580) on an EnVisionMultilabel Reader (Perkin Elmer).

The ISVDs shown in Table 7 inhibit IL-13-induced TF1 proliferation.

TABLE 7 Overview of IC50 values of anti-IL-13 monovalent ISVDs inIL-13-induced TF-1 proliferation assay. compound IC50 (M) F0107004B022.7E−08 F0107004B06 1.0E−05 anti-hIL-13 reference mAb2 4.1E−10anti-hIL-13 reference mAb3 9.2E−11 anti-hIL-13 reference mAb1 2.2E−11

The blocking potency of the anti-TSLP ISVDs was determined in acell-based assay monitoring TSLP mediated proliferation of BaF3 cellstransfected with plasmids encoding hTSLPR and hIL7Ra. Cells were seededat a density of 20.000 cells/well in RPMI 1640 growth medium in cellculture treated white 96 well plates. A dilution series of anti-TSLP

ISVDs were added, followed by addition of 50 pM hTLSP-Fc or 50 pM cynoTSLP-Fc for stimulation of the cells. The human and cyno TSLP sequencesare known (Uniprot accession Uniprot accession Q969D9 and NCBI RefSeqXP_005557555.1, respectively). Recombinant protein was used to performthe assay.

After incubation for 72 hours, cell density and viability was monitoredusing the CellTiter-Glo® Luminescent Cell Viability Assay (Promega,G7571/G7572/G7573) and read-out on an EnVision Multilabel Reader (PerkinElmer). Results are shown in Table 8.

TABLE 8 Potency and efficacy of anti-TSLP monovalent ISVDs inTSLP-induced BaF3 cell proliferation assay. IC50 (M) % inhibition IC50(M) cyno % inhibition ISVD ID hTSLP-Fc hTSLP-Fc TSLP-Fc cyno TSLP-FcF0107501A02 >1.0E−06 46 >1.0E−06 10 F0107529F10 ND ND ND ND ND = notdetermined

6.1.8 Example 8: Binding Affinity of Purified Anti-IL-13 and Anti-TSLPISVDs to Human and Cyno IL-13 and TSLP

Full binding kinetic study by SPR was performed on a BIAcore T100instrument (GE Healthcare).

For IL-13, around 2000 RU of hIL13-Fc or 4000 RU cyno IL13-Fc wasimmobilized directly on a CM5 sensor chip. The ISVDs were then injectedat different concentrations (between 3 μM and 12 nM) for 120s andallowed to dissociate for 900s. Regeneration of the hIL-13-Fc and cynoIL-13-Fc surfaces were performed using a 47s injection of 0.85%H₃PO₄:MilliQ (1:1).

For TSLP, around 8000 RU of anti-hulgG antibody (GE Healthcare) wasimmobilized directly on a CM5 sensor chip. hTSLP-Fc (at 1 μg/mL) or cynoTSLP-Fc (at 0.75 μg/mL) were floated and captured for 120s over thechip. The ISVDs were then injected at different concentrations (between0.4 nM and 3000 nM) for 120 s and allowed to dissociate for 900 s.

Evaluation of the binding curves was done using BIAcore T100 Evaluationsoftware V2.0.3. Kinetic analysis was performed by fitting a 1:1interaction model (Langmuir binding) (Rmax=global; RI=constant=0,offset=0). Interactions which could not meet the acceptance criteria forthe 1:1 interaction model, were fitted using the heterogeneous ligandfit model (RI=constant=0, offset=0).

Kinetic data are shown in Table 9 for IL-13 ISVDs and in Table 10 forTSLP ISVDs.

TABLE 9 KD determination of monovalent anti-IL13 ISVDs via SPR. % majorinteraction k_(a) Model used for fitting Sample Surface present (1/Ms)k_(d) (1/s) K_(D) (M) of binding curves F0107004B02 hIL13-Fc 80 2.2E+042.4E−02 1.1E−06 heterogeneous ligand fit cyno IL13- 81 1.8E+04 3.4E−021.9E−06 heterogeneous ligand Fc fit

TABLE 10 KD determination of monovalent anti-TSLP ISVDs via SPR.hTSLP-Fc cyno TSLP-Fc k_(a) k_(d) K_(D) k_(a) k_(d) K_(D) ID (1/Ms)(1/s) (M) (1/Ms) (1/s) (M) F0107501A02 1.1E+05 5.7E−05 5.1E−10 7.3E+041.8E−03 2.5E−08 F0107529F10 1.2E+07 4.2E−04 3.5E−11 8.7E+05 1.5E−031.7E−09

6.2 Generation of Monospecific Multivalent Polypeptides Binding to IL-13and TSLP, Respectively

6.2.1 Example 10: Generation and In Vitro Characterization of Wild-TypeAnti-IL-13 Bivalent or Biparatopic ISVD Constructs

The selected anti-IL-13 ISVDs (F0107004B02 and F0107004B06) wereformatted into biparatopic and bivalent ISVD constructs. The buildingblocks in the constructs are genetically linked by a flexible 35GS(GlySer) linker. ISVDs were expressed as FLAG3-HIS6-tagged protein inPichia pastoris (amino acid sequences are shown in Table 14). Inductionof ISVD construct expression occurred by stepwise addition of methanol.Clarified medium with secreted ISVD construct was used as startingmaterial for immobilized metal affinity chromatography (IMAC) followedby desalting resulting in 90% purity as assessed by SDS-PAGE.

The biparatopic and bivalent IL-13 constructs were characterised in thebinary and ternary complex blocking AlphaScreen assays (as described inexample 6) as well as in the TF-1 proliferation assay (as described inexample 7). An overview of the generated constructs and their blockingpotencies in the binary and ternary AlphaScreen assay and in the TF-1proliferation assay is shown in Table 11. The biparatopic constructdisplays excellent potencies against hIL-13 and cyIL-13, similar to thepotency of anti-hIL-13 reference mAb1. Also bivalent constructs improvein potency, but not to the same extent as the biparatopic constructF010700029 in the TF1 proliferation assay. In addition, the bivalentconstructs do not reach full inhibition in the IL13-IL13Rα1 Alphascreen.

TABLE 11 Overview of potency of different anti-IL-13 constructs in theAlphascreen assays and of potency and efficacy in the TF-1 proliferationassay. TF1 proliferation IL13-IL13Rα1 IL13-IL13Rα1-IL4Rα assay IC50 (M)AlphaScreen AlphaScreen 200 pM ISVD ISVD construct % % 200 pM cynoconstruct ID description IC50 (M) inhibition IC50 (M) inhibition hIL13IL13 F010700003 F0107004B06- 1.3E−10 99 3.40E−09 100 1.20E−08 ND35GS-F0107004B02 F010700014 F0107004B02- 2.7E−10 84 4.80E−09  989.40E−08 ND 35GS-F0107004B02 F010700029 F0107004B02- 3.1E−10 99 3.00E−09100 2.00E−11 2.50E−10 35GS-F0107004B06 F010700031 F0107004B06- No fitpartial 6.60E−10  99 1.00E−07 ND 35GS-F0107004B06 anti-hIL-13 ND ND NDND 1.50E−11 1.30E−10 reference mAb1 ND = not determined

The most potent anti-IL-13 biparatopic ISVD construct was tested in theIL-13 induced A549 eotaxin release assay. To this end A549 suspensioncells were cultured in Ham's F12K medium supplemented with 10% FCS.Cells were seeded into a 96 well plate at 200.000 cells/well. The nextday 200 pM hIL-13 (Sino Biological cat nr 10369-HNAC) or cyno IL-13(Sino Biological cat nr 11057-CNAH) were added, followed by a dilutionseries of the ISVD constructs. After 24h, Eotaxin-3 was determined inthe supernatants using the MSD ELISA (Human Eotaxin-3 Tissue Culture Kit(Meso Scale, K151ABB-1)). Results are shown in Table 12.

TABLE 12 Potency of F010700029 in the IL-13-mediated Aa549 eotaxinrelease assay. ISVD construct IC50 (M) IC50 (M) ISVD construct IDdescription hIL-13 cyno IL-13 F010700029 F0107004B02-35GS- 2.10E−101.50E−10 F0107004B06 anti-hIL-13 8.00E−11 7.00E−11 reference mAb1Inhibition was 100%.

Competition ELISA was used to test if anti-IL-13 monovalent andbiparatopic ISVD constructs inhibit binding of hIL-13 to IL13Rα2.IL13Rα2 (SinoBiological cat nr 10350-H08H) was coated on a 384 wellSpectraplate (Perkin Elmer). hIL13-Fc (SinoBiological, cat nr10369-H01H) was mixed with serial dilutions of ISVD constructs orpositive control compound IL-13Rα2 and incubated for 1 hour. Afterwashing and blocking, the coated receptor was incubated with the IL13-FcISVD/control mix and incubated for 1 hour. After washing, the presenceof bound IL-13-Fc was detected using anti-human IgG-peroxidase antibodyand a subsequent enzymatic reaction in the presence of substrate esTMB.In the case the ISVD inhibits the IL-13-IL-13Rα2 interaction, detectionof IL-13-Fc disappears in a dose dependent manner. Results are shown inTable 13.

TABLE 13 capacity of anti-IL-13 monovalent and biparatopic ISVDconstructs to inhibit IL-13-IL-13Rα2 complex formation in a competitionELISA % inhibition of construct IL13-IL13Rα2 Compound descriptioncomplex formation IC50 (M) F010704B02 — 37 4.9E−08 F010704B06 — 49 Nofit F027100271 F0107004B02-9GS- 53 No fit ALB23002-9GS- F0107004B06IL-13Rα2 — 100 7.9E−09

TABLE 14 Amino acids sequences of wild-type anti-IL13 biparatopic and bivalent ISVD constructs. ISVD  ISVD  construct construct ID description Sequence F010700003 F0107004B06-EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID  35GS- GFTFNNYAMKWVRQAPGKGLEWVSSNO: 148) F0107004B02 ITTGGGSTDYADSVKGRFTISRDNR KNTLYLQMNSLKPEDTAVYYCANVPFGYYSEHFSGLSFDYRGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQA GGSLRLSCAASGRTFSSYRMGWFRQAPGKEREFVAALSGDGYSTYTANSV NSRFTISRDNAKNTVYLQMNSLKPEDTAIYYCAAKLQYVSGWSYDYPYWG QGTLVTVSS F010700014 F0107004B02-EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID  35GS- GRTFSSYRMGWFRQAPGKEREFVAANO: 149) F0107004B02 LSGDGYSTYTANSVNSRFTISRDNA KNTVYLQMNSLKPEDTAIYYCAAKLLQYVSGWSYDYPYWGQGTLVTVSSG GGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGG SLRSCAASGRTFSSYRMGWFRQAPGKEREFVAALSGDGYSTYTANSVNSR FTISRDNAKNTVYLQMNSLKPEDTAIYYCAAKLQYVSGWSYDYPYWGQGT LVTVSS F010700029 F0107004B02-EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID  35GS- GRTFSSYRMGWFRQAPGKEREFVAANO: 150) F0107004B06 LSGDGYSTYTANSVNSRFTISRDNA KNTVYLQMNSLKPEDTAIYYCAAKLQYVSGWSYDYPYWGQGTLVTVSSGG GGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS LRLSCAASGFTFNNYAMKWVRQAPGKGLEWVSSITTGGGSTDYADSVKGR FTISRDNRKNTLYLQMNSLKPEDTAVYYCANVPFGYYSEHFSGLSFDYRG QGTLVTVSS F010700031 F0107004B06-EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID  35GS- GFTFNNYAMKWVRQAPGKGLEWVSSNO: 151) F0107004B06 ITTGGGSTDYADSVKGRFTISRDNR KNTLYLQMNSLKPEDTAVYYCANVPFGYYSEHFSGLSFDYRGQGTLVTVS SGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQP GGSLRLSCAASGFTFNNYAMKWVRQAPGKGLEWVSSITTGGGSTDYADSV KGRFTISRDNRKNTLYLQMNSLKPEDTAVYYCANVPFGYYSEHFSGLSFD YRGQGTLVTVSS

6.2.2 Example 11: Generation and Characterization of Wild-TypeBiparatopic Anti-TSLP ISVD Constructs

The selected TSLPR blockers (F0107501A02 and F0107529F10) were combinedinto biparatopic ISVD constructs. The constructs were expressed asFLAG3-HIS6-tagged protein in Pichia pastoris (amino acid sequences areshown in Table 16). Induction of ISVD construct expression occurred bystepwise addition of methanol. Clarified medium with secreted ISVDconstruct was used as starting material for immobilized metal affinitychromatography (IMAC) followed by desalting resulting in 90% purity asassessed by SDS-PAGE.

The biparatopic constructs were titrated as purified proteins against 50or 5 pM hTSLP and 50 or 5 pM cyno TSLP in the BaF3 proliferation assay(as described in example 7) and compared to different anti-TSLPreference compounds (anti-hTSLP reference mAb2 and anti-hTSLP referencemAb1). The data is summarized in Table 15 (hTSLP and cyno TSLP). Thebiparatopic constructs clearly outperform the reference mAbs. Thebiparatopic construct with F0107501A02 at the N-terminus shows improvedreactivity towards cyno TSLP compared to the construct with F0107501A02at the C-terminus.

TABLE 15 Overview of IC50 values (M) of different biparatopic anti-TSLPformats in the human and cyno TSLP, respectively, mediated BaF3proliferation assay. IC50 (M) IC50 (M) Conc TSLP used for ISVD constructID ISVD construct description hTSP cyno TSLP stimulation (pM) F010703842F0107529F10-35GS-F0107501A02 4.8E−11 3.9E−09 50 F027400016 F0107501A02(E1D, L11V, A14P, E16G, 4.6E−12 6.0E−11 5 A41P, I43K, E44Q, A74S, E75K,M78L, K83R, A84P, G88A, V89L)*-35GS-529F10 anti-hTSLP 1.6E−10 >1.0E−07 50 reference mAb2 anti-hTSLP 5.5E−10 3.8E−09 5 reference mAb1 *( )indicates the amino acid substitutions introduced into the (parental)monovalent building block.

TABLE 16 Amino acids sequences of biparatopic anti-TSLP ISVD constructs F010703842 and F027400016. ISVD ISVD constructconstruct ID description Sequence F010703842 F0107529F10-EVQLVESGGGLVQAGGSLRLSCAAS (SEQ ID  35GS- GFTFADYDYDIGWFRQAPGKEREGVNO: 152) F0107501A02 SCISNRDGSTYYTDSVKGRFTISSD NAKNTVSLQMNSLKPEDTAVYYCAVEIHCDDYGVENFDFDPWGQGTLVTV SSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQ AGESLRLSCAASGSGFGVNILYWYRQAAGIERELIASITSGGITNYVDSV KGRFTISRDNAENTMYLQMNSLKAEDTGVYYCASRNIFDGTTEWGQGTLV TVSS F027400016 F0107501A02 DVQLVESGGGVVQPGGSLRLSCAAS (SEQ ID  (E1D, L11V, GSGFGVNILYWYRQAPGKQRELIAS NO: 153) A14P, E16G, ITSGGITNYVDSVKGRFTISRDNSK A41P, I43K,  NTLYLQMNSLRPEDTALYYCASRNIE44Q, A74S,  FDGTTEWGQGTLVTVSSGGGGSGGG E75K, M78L, GSGGGGSGGGGSGGGGSGGGGSGGG K83R, A84P,  GSEVQLVESGGGVVQPGGSLRLSCA G88A, ASGFTFADYDYDIGWFRQAPGKERE V89L)*- GVSCISNRDGSTYYADSVKGRFTIS 35GS-529F10RDNSKNTVYLQMNSLRPEDTALYYC AVEIHCDDYGVENFDFDPWGQGTLV TVSS *( ) indicatesthe amino acid substitutions introduced into the (parental) monovalentbuilding block.

6.3 Sequence Optimization of the Anti-IL-13 and Anti-TSLP MonovalentISVDs

6.3.1 Example 12: Sequence Optimization of Anti-IL-13 and Anti-TSLPMonovalent ISVDs

Anti-IL-13 ISVDs F0107004B02 and F0107004B06 and anti-TSLP ISVDsF0107501A02 and F0107529F10 were further sequence optimized.

Sequence optimization involves replacing one or more specific amino acidresidues in the sequence in order to improve one or more (desired)properties of the ISVDs.

Some examples of such sequence optimization are mentioned in the furtherdescription herein and for example include, without limitation:

-   1) Substitutions in parental wild type ISVD sequences to yield ISVD    sequences that are more identical to the human VH3-JH germline    consensus sequences, a process called humanization. To this end,    specific amino acids, with the exception of the so-called hallmark    residues, in the FRs that differ between the ISVD and the human    VH3-JH germline consensus are altered to the human counterpart in    such a way that the protein structure, activity and stability are    kept intact.-   2) Substitutions towards the llama germline to increase the    stability of the ISVD, which is defined as camelisation. To this    end, the parental wild type ISVD amino acid sequence is aligned to    the llama IGHV germline amino acid sequence of the ISVD (identified    as the top hit from a BlastP analysis of the ISVD against the llama    IGHV germlines).-   3) Substitutions that improve long-term stability or properties    under storage, substitutions that increase expression levels in a    desired host cell or host organism, and/or substitutions that remove    or reduce (undesired) post-translational modification(s) (such as    glycosylation or phosphorylation), again depending on the desired    host cell or host organism. To avoid N-terminal pyroglutamate    formation, standardly an E1D mutation is introduced in the    N-terminal building block of a multivalent Nb, without impact on    potency or stability. During sequence optimization of the building    blocks, the E1D mutation is therefore not consistently introduced.-   4) Mutations on position 11 towards Val and on position 89 towards    Leu to minimize the binding of any naturally occurring pre-existing    antibody activity.

F0107004602 and F0107004606

Sequence optimisation of anti-IL-13 ISVD F0107004B02 resulted in a finalsequence optimised variant F027100019, which comprises 8 amino acidsubstitutions (i.e. E1D, Lily, A14P, N64K, S65G, A74S, K83R, I89L)compared to the parental ISVD F107004B02. Sequence optimisation ofanti-IL-13 ISVD F0107004B06 resulted in a final sequence optimisedvariant F027100183, which comprises 4 amino acid substitutions (i.e.Lily, R74S, K83R, V89L) compared to the parental ISVD F107004B06.

The sequence optimised variants were assembled from oligonucleotidesusing a PCR overlap extension method. The variants were expressed in E.coli and purified by IMAC and desalting. F027100019 and F027100183 wereevaluated for their hIL-13 binding capacity by surface plasmonresonance, using hIL-13 from Peprotech (cat nr 200-13). Additionally,F027100019 was tested for its neutralizing activity in the eotaxinrelease assay. Monomeric behavior of both variants was monitored by SizeExclusion-HPLC (SE-HPLC). Thermal stability of the variants was testedin a thermal shift assay (TSA) using the Lightcycler (Roche). In thisassay, the parental ISVDs and their variants are incubated at differentpH's in the presence of sypro orange and a temperature gradient isapplied. When the ISVDs start denaturing, sypro orange binds and themeasured fluorescence increases suddenly, as such a melting temperaturecan be determined for a certain pH. Results are summarized in Table 17and Table 18.

TABLE 17 results of the analysis of the sequence optimization variantF027100019 of anti-IL-13 ISVD F0107004B02. IC50 (nM) Eotaxin Kon Koff KDTm (° C.) SE-HPLC release hIL-13 hIL-13 hIL-13 at pH 7 % main ISVD IDMutation(s) assay (1/Ms) (1/s) (M) TSA peak F0107004B02 WT nd 2.20E+2.40E− 1.10E− 67  94 04* 02* 06* F027100019 E1D, L11V, 47 3.50E+ 1.20E−3.40E− 70 100 A14P, N64K, 04 03 08 S65G, A74S, K83R, I89L nd = notdetermined, *measured on hIL-13-Fc

F027100019 exhibited good potency in the Eotaxin release assay and itsaffinity to hIL-13 was determined to be 34 nM in SPR. The Tm ofF027100019 is 3° C. higher than for the parental ISVD F0107004B2. The %framework identity in the framework regions for F027100019 is 85% basedon the AbM definition (see Antibody Engineering, Volt by Kontermann &Dübel (Eds), Springer Verlag Heidelberg Berlin, 2010) and 86% based onthe Kabat definition.

TABLE 18 results of the analysis of the sequence optimization variant ofanti-IL13 ISVD F0107004B06. Kon Koff KD hIL-13 hIL-13 hIL-13 Tm (° C.)at SE-HPLC % ISVD ID Mutation(s) (1/Ms) (1/s) (M) pH 7 TSA main peakF0107004B06 WT 2.50E+05 3.30E−03 2.60E−08 nd nd F027100183 L11V, R74S,2.80E+05 7.50E−03 2.70E−08 61 100 K83R, V89L Nd = not determined

Affinity of F027100183 is similar compared to the WT sequence and thevariant has a Tm of 61° C. The variant elutes as a 100% monomeric peakon SE-HPLC. The % framework identity in the framework regions forF027100183 is 94.4% based on the AbM definition and 93.1% based on theKabat definition.

F0107529F10

Sequence optimisation of anti-TSLP ISVD F0107529F10 resulted in a finalsequence optimised variant F027400021, which comprises 8 amino acidsubstitutions (i.e., Lily, A14P, T60A, S71R, A74S, S79Y, K83R, V89L)compared to the parental ISVD F0107529F10. Sequence optimisation ofanti-TSLP ISVD F0107501A02 resulted in a final sequence optimisedvariant F027400160, which comprises 6 amino acid substitutions (i.e.,Lily, A14P, E16G, A74S, K83R, V89L) compared to the parental ISVDF0107501A02.

The sequence optimised variants were assembled from oligonucleotidesusing a PCR overlap extension method. The constructs were expressed inE. coli and purified by IMAC and desalting. The variants were evaluatedfor their binding capacity to human and cyno TSLP by surface plasmonresonance. Monomeric behaviour of all variants was monitored by SizeExclusion-HPLC (SE-HPLC) and the thermal stability in a thermal shiftassay (TSA). Additionally, the variants of F0107501A02 were tested fortheir blocking activity on hTSLP in the ternary complex Alphascreen.Results are summarized in Table 19 and Table 20.

TABLE 19 Results of the analysis of the sequence optimization variantsof anti-TSLP ISVD F0107529F10, koff K_(D) koff cyno hTSLP- K_(D) cyno Tm(° C.) SE-HPLC hTSLP TSLP hFc TSLP-hFc at pH 7 % main ISVD ID Mutations(s-1) (s-1) (M) (M) TSA peak F0107529F10 WT 6.60E−05 1.80E−03 3.50E−111.70E−09 64.0 nd F010704099 A14P, T60A, S71R, 7.70E−05 1.80E−03 3.80E−113.30E−09 75.4 100 A74S, S79Y, K83R F027400021 L11V, A14P, T60A, nd nd ndnd 74.0 100 S71R, A74S, S79Y, K83R, V89L nd = not determined

Intermediate variant F010704099 with mutations A14P, T60A, S71R, A74S,S79Y, K83R showed a similar affinity on hTSLP and a 2-fold loweraffinity on cyno TSLP compared to the parental ISVD F0107529F10. The Tmshowed an overall increase of 11.4° C. compared to the parental ISVD.Two additional mutations, i.e. L11V and V89L, were introduced intovariant F010704099 to minimize pre-existing antibody binding, resultingin the final variant F027400021. The % framework identity in theframework regions for F027400021 is 89.9% based on the AbM definitionand 88.5% based on the Kabat definition.

TABLE 20 Results of the analysis of the sequence optimization (SO)variants of anti-TSLP ISVD F0107501A02. Ternary Koff koff complex hTSLPcyno TSLP AlphaScreen Tm (° C.) at SE-HPLC % ISVD ID Mutation(s) (s-1)(s-1) IC50 (M) pH 7 TSA main peak F0107501A02 WT 5.4E−05 1.5E−03 2.8E−1068.5 100 F010704076 A14P, E16G, 6.2E−05 1.6E−03 2.7E−10 70.8 99.3 A74S,K83R F027400160 L11V, A14P, 1.2E−04 4.3e−03 nd 70.0 100 E16G, A74S,K83R, V89L

Intermediate variant F010704076 with mutations A14P, E16G, A74S, K83Rshowed a similar off-rate on hTSLP and on cyno TSLP compared to theparental ISVD F0107501A02 and a similar potency in ternary complexAlphascreen. The Tm showed an overall increase of 12.3° C. compared tothe parental ISVD. Two additional mutations i.e., L11V and V89L, wereintroduced into variant F010704076 to minimize pre-existing antibodybinding, resulting in the final variant F027400160.

The % framework identity in the framework regions for F027400160 is83.1% based on the AbM definition and 80.5% based on the Kabatdefinition.

TABLE 21 Amino acid sequences of  SO version of ISVD F0107004B02, F0107004B06, F0107501A02 and F107529F10. ISVD  ISVD ID descriptionSequence F010704076 F0107501A02 EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID(A14P, E16G, GSGFGVNILYWYRQAAGIERELIAS NO: 160) A74S, K83R)*ITSGGITNYVDSVKGRFTISRDNSE NTMYLQMNSLRAEDTGVYYCASRNI FDGTTEWGQGTLVTVSSF010704099 F0107529F10  EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID (A14P, T60A,GFTFADYDYDIGWFRQAPGKEREGV NO: 161) S71R, A74S, SCISNRDGSTYYADSVKGRFTISRDS79Y, K83R)* NSKNTVYLQMNSLRPEDTAVYYCAV EIHCDDYGVENFDFDPWGQGTLVTV SSF027100019 F0107004B02  DVQLVESGGGVVQPGGSLRLSCAAS (SEQ ID (E1D, L11V,GRTFSSYRMGWFRQAPGKEREFVAA NO: 162) A14P, N64K, LSGDGYSTYTANSVKGRFTISRDNSS65G, A74S, KNTVYLQMNSLRPEDTALYYCAAKL K83R, I89L)*QYVSGWSYDYPYWGQGTLVTVSSAA A F027100183 F0107004B06EVQLVESGGGVVQPGGSLRLSCAAS (SEQ ID (L11V, R74S, GFTFNNYAMKWVRQAPGKGLEWVSSNO: 163) K83R, V89L)* ITTGGGSTDYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTALYYCANVP FGYYSEHFSGLSFDYRGQGTLVTVS S F027400021F0107529F10  EVQLVESGGGVVQPGGSLRLSCAAS (SEQ ID (L11V, A14P,GFTFADYDYDIGWFRQAPGKEREGV NO: 164) T60A, S71R, SCISNRDGSTYYADSVKGRFTISRD A74S, S79Y,  NSKNTVYLQMNSLRPEDTALYYCAVK83R, V89L)* EIHCDDYGVENFDFDPWGQGTLVTV SS F027400160 F0107501A02EVQLVESGGGVVQPGGSLRLSCAAS (SEQ ID (L11V, A14P, GSGFGVNILYWYRQAAGIERELIASNO: 165) E16G, A74S,  ITSGGITNYVDSVKGRFTISRDNSE K83R, V89L)*NTMYLQMNSLRAEDTGLYYCASRNI FDGTTEWGQGTLVTVSS *( ) indicates the aminoacid substitutions introduced into the (parental) monovalent buildingblock.

6.4 Multispecific ISVD Construct F027400161

The above identified optimized ISVDs F027100019 (optimized variant ofF0107004802), F027100183 (optimized variant of F0107004806), F027400021(optimized variant of F0107529F10), and F027400160 (optimized version ofF0107501A02) were used for the generation of multispecific ISVDconstruct F027400161. The optimized monovalent building blocks used inF027400161 are designated in the following in an abbreviated formaccording to their ISVD origin as 04B02, 04B06, 529F10, and 501A02,respectively.

6.4.1 Example 15: Multispecific ISVD Construct Generation

Identification of ISVD-containing polypeptide F027400161 (SEQ ID NO: 1)binding to IL-13 and TSLP resulted from a data-driven bispecificengineering and formatting campaign in which several anti-TLSP buildingblocks, several anti-IL13 building blocks and the anti-HSA buildingblock ALB23002 were included. Different positions/orientations of thebuilding blocks and different linker lengths (9GS vs 35GS) were appliedand proofed to be critical for different parameters (potency,cross-reactivity, expression yield, etc.).

A large panel of different ISVD constructs was transformed in PichiaPastoris for small scale productions. Induction of ISVD expressionoccurred by stepwise addition of methanol. Clarified medium withsecreted ISVD was used as starting material for purification via ProteinA affinity chromatography followed by desalting. The purified sampleswere used for functional characterisation and expression evaluation.

Some constructs showed impaired potencies depending on linker length andrelative position of ISVD building blocks. For example: potency for cynoTSLP of the anti-TSLP biparatopic combinations 501A02-529F10 is stronglyimpaired when linked with a short 9GS linker. Some constructs showed lowexpression levels depending on the combination and order of the buildingblocks. For the bispecifics comprising 4B02-4B06 expression was best incombination with 501A02-529F10.

TABLE 22 Selection of different multispecific ISVD formats evaluated.linker linker linker linker ISVD ID BB1 1 BB2 2 BB3 3 BB4 4 BB5F027400161 4B02 35 GS 4B06  9 GS 501A02  9 GS ALB  9 GS 529F10F027400162 4B02  9 GS 501A02  9 GS 4B06  9 GS 529F10  9 GS ALBF027400163 501A02  9 GS ALB  9 GS 529F10  9 GS 4B02 35 GS 4B06F027400183 501A02 35 GS 529F10  9 GS 4B02  9 GS ALB  9 GS 4B06F027400189 4B02  9 GS ALB  9 GS 4B06  9 GS 501A02 35 GS 529F10F027400283 501A02  9 GS 4B02  9 GS 529F10  9 GS 4B06 35 GS ALBF027400284 501A02 35 GS 4B02 35 GS 529F10 35 GS 4B06 35 GS ALBF027400296 4B02  9 GS 501A02  9 GS 4B06  9 GS 529F10  9 GS ALB (N73Q)*F027400298 501A02  9 GS 4B02  9 GS 529F10  9 GS 4B06  9 GS ALB (N73Q)**( )indicates the amino acid substitutions introduced into the(parental) monovalent building block. BB = building block, ALB =ALB23002

Table 23 illustrates that different yields ranging from low to hightiter were obtained for six constructs comprising the same buildingblocks but ordered in different ways and connected with different linkerlengths. Highest expression titers are obtained for constructscomprising the IL-13 ISVDs 4B02 and 4B06 linked via a 35GS linker(F027400161 and F027400163). In addition, the solubility of F027400161and F027400163 was much higher than their respective counterpartsF027400296 and 298, of which building blocks are linked with four 9GSlinkers and ALB is positioned at the C-terminus.

Subsequently, the large bispecific panel was trimmed down to a panel of2 bispecific constructs, consisting of ISVD constructs F027400161 andF027400163 proven to be potent on both targets (human and cyno) andhaving the potential of high expression levels, based on preliminaryyield estimates.

Larger scale 2 L and 5 L productions in Pichia Pastoris were done forexpression yield determination and assessment of biophysical propertiesand pre-existing antibody reactivity.

Table 24 and example 22 demonstrate that pre-existing antibodyreactivity is driven by the orientation of the building blocks and thelinker lengths.

TABLE 23 Expression levels and solubility of 6 ISVD constructs with thebuilding blocks of 4B02, 4B06, 501A02 and 529F10 in differentorientations and/or with different linker lengths. Yield 5 ml Yield 5 LISVD construct expression fermentation solubility ID BB1 linker 1 BB2linker 2 BB3 linker 3 BB4 linker 4 BB5 (μg/ml) (g/L) (mg/ml) F0274001614B02 35 GS 4B06 9 GS 501A02 9 GS ALB  9 GS 529F10 124 4.7 145 F027400163501A02  9 GS ALB 9 GS 529F10 9 GS 4B02 35 GS 4B06 114 4.4 >150F027400189 4B02  9 GS ALB 9 GS 4B06 9 GS 501A02 35 GS 529F10 77F027400183 501A02 35 GS 529F10 9 GS 4B02 9 GS ALB  9 GS 4B06 55F027400296 4B02  9 GS 501A02 9 GS 4B06 9 GS 529F10  9 GS ALB 80 2.4 Verylow (N73Q)* F027400298 501A02  9 GS 4B02 9 GS 529F10 9 GS 4B06  9 GS ALB80 2.4 25 (N73Q)* *( )indicates the amino acid substitutions introducedinto the (parental) monovalent building block.

TABLE 24 Binding of pre-existing antibodies present in 96 human serumsamples to F027400161, F027400163 and F027400164 compared to controlISVD construct F027301186. 25% Median 75% Construct ID BB1 linker 1 BB2linker 2 BB3 linker 3 BB4 linker 4 BB5 percentile RU levels percentileF027301186 1E07 35 GS 1E07 35 GS 1C02 9 GS ALB  9 GS 1C02 61 135 622F027400161 4B02 35 GS 4B06  9 GS 501A02 9 GS ALB  9 GS 529F10-A 8 22 34F027400163 501A02  9 GS ALB  9 GS 529F10 9 GS 4B02 35 GS 4B06-A 19 34 63F027400164 501A02  9 GS 4B02  9 GS 529F10 9 GS 4B06  9 GS ALB-A 4 13 24

Finally, ISVD construct F027400161 was selected based on potency,reduced binding to pre-existing antibodies and superior expressionlevels and CMC characteristics

6.4.2 Example 16: Multispecific ISVD Construct Binding Affinity to TSLP,IL13 and Serum Albumin

The affinity, expressed as the equilibrium dissociation constant(K_(D)), of F027400161 towards human and cyno TSLP (Human and cyno TSLPsequences are known (Uniprot accession Uniprot accession Q969D9 and NCBIRefSeq XP_005557555.1, respectively). Recombinant protein was used toperform the assay, human IL-13 (Sino Biological cat nr 10369-HNAC), cynoIL-13 (Sino Biological cat nr 11057-CNAH) and rhesus IL-13 (R&D systemscat nr 2674-RM) and human (Sigma Aldrich, cat nr A8763), cyno and mouse(Albumin Bioscience cat nr N1204H1CM) serum albumin was quantified bymeans of in-solution affinity measurements on a Gyrolab xP Workstation(Gyros).

Under K_(D)-controlled measurements a serial dilution of TSLP or IL-13(ranging from 1 μM-0.25 fM) or serum albumin (ranging from 100 μM-320pM) and a fixed amount of F027400161 (5 pM or 10 pM in case of TSLP andIL13 and 20 nM in case of serum albumin) were mixed to allow interactionand incubated for either 48 or 72 hours (in case of TSLP and IL13) or 2hours (in case of serum albumin) to reach equilibrium.

Under receptor-controlled measurements a serial dilution of TSLP orIL-13 (ranging from 1 μM-0.25 fM) or serum albumin (ranging from 100μM-320 pM) and a fixed amount of F027400161 (250 pM in case of TSLP, 10nM in case of IL-13 and 1 μM in case of serum albumin) were mixed toallow interaction and incubated for either 48 or 72 hours (in case ofTSLP and IL-13) or 2 hours (in case of serum albumin) to reachequilibrium.

Biotinylated human TSLP/IL-13/serum albumin was captured in themicrostructures of a Gyrolab Bioaffy 1000 CD, which contains columns ofbeads and is used as a molecular probe to capture free F027400161 fromthe equilibrated solution. The mixture of TLSP/IL-13/serum albumin andF027400161 (containing free TLSP/IL-13/serum albumin, free F027400161and TLSP/IL-13/serum albumin-F027400161 complexes) was allowed to flowthrough the beads, and a small percentage of free F027400161 wascaptured, which is proportional to the free ISVD concentration. Afluorescently labeled anti-V_(HH) antibody, ABH0086-Alexa647, was theninjected to label any captured F027400161 and after rinsing away excessof fluorescent probe, the change in fluorescence was determined. Fittingof the dilution series was done using Gyrolab Analysis software, whereK_(D)- and receptor-controlled curves were analyzed to determine theK_(D) value.

The results (Table 25) demonstrate that the multispecific ISVD constructbinds human/cyno TSLP and human/cyno/rhesus IL13 with high affinity.

TABLE 25 Binding affinities of F027400161 to human, rhesus and cyno TSLPand IL13 and human, cyno and mouse serum albumin (sequences of cyno andrhesus TSLP and cyno and rhesus albumin are identical). human cynomolgusmonkey rhesus monkey 95% Cl 95% Cl 95% Cl Incubation Antigen K_(D) (pM)(pM) K_(D) (pM) (pM) K_(D) (pM) (pM) time (h) TSLP 1.8 1.2- 55.9 51-6148 2.3 2.3 1.4- 44.0 26-62 48 3.3 1.0 0.65- 59.0 35-83 72 1.4 IL13 4.23.6- 3.8 2.8- 6.4 5.3- 48 4.8 4.9 7.4 6.3 5.3- 1.5 0.7- 1.9 0.6- 48 7.32.3 3.1 4.7 3.8- 3.5 2.7- 1.5 0.5- 72 5.7 4.2 2.4 human cynomolgusmonkey mouse 95% Cl 95% Cl 95% Cl Incubation Antigen K_(D) (nM) (nM)K_(D) (nM) (nM) K_(D) (nM) (nM) time (h) SA 119 99.4- 198 185- 18001700- 2 139 211 1900 110 56- 133 70- 2 162 196 145 69- 145 78- 2 222 213

6.4.3 Example 17: Multispecific ISVD Construct Binds Selectively to TSLPand IL13

Absence of F027400161 binding to IL-4 and IL-7 as IL13 and TSLP relatedcytokines was assessed via SPR (Proteon XPR36), respectively.

Cytokines were immobilized on a Proteon GLC sensor chip at 25 μg/mL for600s using amine coupling, with 80 seconds injection of EDC/NHS foractivation and a 150 seconds injection of 1 M Ethanolamine HCl fordeactivation (ProteOn Amine Coupling Kit. cat. 176-2410). Flow rateduring activation and deactivation was set to 30 μl/min and duringligand injection to 25 μl/min. The pH of the 10 mM Acetateimmobilization buffer was 6.0 for IL13 and IL4 (Peprotech cat nr 200-07)and 5.5 for TSLP and IL7 (R&D systems cat nr 204-IL/CF).

Next, 1 μM of F027400161 was injected for 2 minutes and allowed todissociate for 600s at a flow rate of 45 μL/min. As running buffer PBS(pH7.4)+0.005% Tween 20 was used. As positive controls, 100 nM α-hIL4 Aband 100 nM α-IL7 Ab were injected. Interaction between F027400161 andthe positive controls with the immobilized targets was measured bydetection of increases in refractory index which occurs as a result ofmass changes on the chip upon binding.

All positive controls did bind to their respective target. No bindingwas detected of F027400161 to human IL4 and IL7.

In addition, it was investigated if F027400161 could bind to the shortform of TSLP. To this end, TSLP and the short form of TSLP (as describedin Fornasa, 2015) were immobilized on a proteon GLC sensor chip at 10μg/mL, respectively 5 μg/ml for 150s using amine coupling as describedabove. The pH of the 10 mM Acetate immobilization buffer was 5.5 forTSLP and 4.0 for the short form of TSLP.

Next, 500 nM of F027400161 was injected for 2 minutes and allowed todissociate for 600s at a flow rate of 45 μL/min. As running buffer PBS(pH7.4)+0.005% Tween 20 was used. As reference compound, 500 nManti-hTSLP reference mAb1 was injected and as positive control 500 nMα-hTSLP pAb (Abcam ab47943).

Whereas the positive control did bind to both the long (normal) andshort form of TSLP, no binding was detected of F027400161 and anti-hTSLPreference mAb1 to the short form of TSLP.

6.4.4 Example 18: Simultaneous Binding of Multispecific ISVD Constructto IL13, TSLP and HSA

A Biacore T200 instalment was used to determine whether F027400161 canbind simultaneously to hTSLP and hIL13. To this end hTSLP (recombinanthuman TSLP is commercially available, such as from R&D Systems (cat nr1398-TS) was immobilized on a CM5 Sensor chip via amine coupling. 100 nMof F027400161 was injected for 2 min at 10 μl/min over the TSLP surfacein order to capture the ISVD construct via the TSLP building blocks501A02-529F10. Subsequently either 100 nM of hIL13 (PeproTech, cat nr200-13), HSA or hOX40L or 1000 nM of HSA were injected or mixtures of100 nM IL13+100 nM HSA, 100 nM IL13+1000 nM HSA, 100 nM OX40L+100 nMHSA, 100 nM OX40L+1000 nM HSA or 100 nM IL13+100 nM OX40L, at a flowrate of 10 μl/min for 2 min followed by a subsequent 300 secondsdissociation step. The TSLP surfaces are regenerated with a 1 minuteinjection of 0.5% SDS+10 mM glycine pH3 at 45 μl/min. The sensorgram(FIG. 1) demonstrates that F027400161 can bind human IL13, human TSLPand HSA simultaneously as shown by the increase in response units aftercapture on TSLP: ^(˜)130 RU increase from IL13 only, ^(˜)60 RU increasefrom 100 nM HSA and ^(˜)350 RU from 1000 nM HSA only, ^(˜)180 RUincrease for the IL13 and 100 nM HSA mixture, and ^(˜)500 RU for theIL13 and 1000 nM HSA mixture. Higher concentrations of HSA were neededto see decent RU increase levels, due to the lower affinity ofF027400161 for HSA (see example 16).

Example 19: Multispecific ISVD Construct Inhibition of IL13 InducedEotaxin Release In Vitro

Functional activity of soluble IL13 from the different species ofinterest (human, rhesus and cynomolgus monkey) and inhibition thereof byF027400161 was studied using a cell based assay investigating eotaxinrelease by A549 human lung carcinoma cells.

To this end, A549 suspension cells were cultured in Ham's F12K medium,supplemented with 10% FCS, and seeded into a 96 well plate at 400.000cells/well. After 24 hours incubation, a dilution series of F027100161or reference compounds (anti-hIL-13 reference mAb1 and anti-hIL-13reference mAb2) were added. After 20 min incubation, human IL13 (SinoBiological cat nr 10369-HNAC), cyno IL13 (Sino Biological cat nr11057-CNAH) or rhesus IL13 (R&D Systems, cat nr 2674-RM-025) is added toa final concentration of 160 pM. After further incubation for 24 hoursin the presence of 30 μM HSA, heparin is added at a final concentrationof 50 μg/ml, to enhance the eotaxin expression. After an additional 4hours of incubation, eotaxin-3, secreted in the cell supernatant wasquantified by use of the Human CCL26/Eotaxin-3 DuoSet ELISA (R&Dsystems, DY346).

F027400161 inhibited human, cyno and rhesus IL13-induced eotaxin-3release in a concentration-dependent manner with an IC50 of 194 pM (forhuman IL13), 1040 pM (for cyno IL13) and 713 pM (for rhesus IL13),comparable to the reference compound anti-hIL-13 reference mAb1, andbetter than the reference compound anti-hIL-13 reference mAb2 (Table 26,FIG. 2).

TABLE 26 IC50 values of F027400161 mediated neutralization of human,cyno and rhesus IL13 in the eotaxin release assay versus the referencecompounds anti-hIL-13 reference mAb1 and anti- hIL-13 reference mAb2.anti-hIL-13 reference anti-hIL-13 reference F027400161 mAb1 mAb2 HumanCyno Rhesus Human Cyno Rhesus Human Cyno Rhesus antigen IL13 IL13 IL13IL13 IL13 IL13 IL13 IL13 IL13 eotaxin 194 1040 713 45 160 99 668 nd 3560release assay (IC50, pM) nd = not determined

6.4.5 Example 20: Multispecific ISVD Construct Inhibition of 1113Induced STAT-6 Activation in HEK-Blue 114/1113 Cells

HEK-Blue™ IL-4/IL-13 cells were generated by stable transfection ofHEK293 cells with the human STAT6 gene and a STAT6-inducible SEAPreporter gene. Upon IL-4 and IL-13 stimulation, the cells produceSTAT6-induced SEAP secreted in the supernatant quantified byQUANTI-Blue™.

HEK-Blue™ cells were cultured DMEM, supplemented with 10% FBS and seededinto a 96 well plate at 50.000 cells/well. A dilution series ofF027100161 or reference compounds (anti-hIL-13 reference mAb1 andanti-hIL-13 reference mAb2) was pre-incubated with 10 pM hIL13 (SinoBiological cat nr 10369-HNAC) or cyno IL-13 (Sino Biological cat nr11057-CNAH) for 1 hour at room temperature and added to the cells. Afterincubation for 22 to 24 hours in the presence of 30 μM HSA, 40 μl of thecell supernatant was mixed with 160 μl QUANTI-Blue™. Secreted SEAP wasquantified by measuring absorption at 620 nm on a Clariostar instrument.

F027400161 inhibited human and cyno IL-13 induced SEAP secretion in aconcentration-dependent manner with an IC50 of 32.8 pM (for human IL-13)and 53.4 pM (for cyno IL-13), better than the reference compoundanti-hIL-13 reference mAb2 (Table 27, FIG. 3).

TABLE 27 IC50 values of F027400161 mediated neutralization of human andcyno IL-13 in the SEAP reporter assay versus the reference compoundsanti-hIL-13 reference mAb1 and anti-hIL-13 reference mAb2. Construct IDIC50 hIL13 (M) IC50 cyno IL13 (M) F027400161 3.28E−11 5.34E−11anti-hIL-13 reference 1.10E−10 4.26E−10 mAb2 anti-hIL-13 reference3.45E−12 5.85E−12 mAb1

6.4.6 Example 21: Multispecific ISVD Construct Inhibition ofTSLP-Induced Ba/F3 Cell Proliferation In Vitro

Functional activity of soluble TSLP from the different species ofinterest (human, rhesus and cynomolgus monkey) and inhibition thereof byF027400161 was studied using a cell-based assay investigatingproliferation of BaF3 cells transfected with plasmids encoding hTSLPRand hIL7Ra.

Cells were seeded at a density of 15000 cells/well in RPMI 1640 growthmedium in cell culture treated white 96 well plates. A dilution seriesof F027100161 or reference compounds (anti-hTSLP reference mAb1) wereadded, followed by addition of 5 pM human or cyno TLSP for stimulationof the cells. The human and cyno TSLP sequences are known (Uniprotaccession Uniprot accession Q969D9 and NCBI RefSeq XP_005557555.1,respectively). Recombinant protein was used to perform the assay. Afterincubation for 48 hours in the presence of 30 μM HSA, cell density andviability were monitored using the CellTiter-Glo® Luminescent CellViability Assay (Promega, G7571/G7572/G7573) and read-out on an EnVisionMultilabel Reader (Perkin Elmer).

F027400161 inhibited human and cyno TSLP dependent proliferation ofBa/F3 cells in a concentration-dependent manner with an IC50 of 7.8 pM(for human TSP) and 24 pM (for cyno TSLP), performing hence much betterthan the reference compound anti-hTSLP reference mAb1 (Table 28, FIG.4).

TABLE 28 IC50 values of F027400161 mediated neutralization of human andcyno TSLP induced BaF3 proliferation versus the reference compoundanti-hTSLP reference mAb1. F027400161 anti-hTSLP reference mAb1 antigenHuman Cyno Human Cyno TSLP TSLP TSLP TSLP BaF3 proliferation 7.8 24 3563180 assay (IC50, pM)

6.4.7 Example 22: Multispecific ISVD Construct Binding to Pre-ExistingAntibodies

The pre-existing antibody reactivity of ISVD construct F027400161 wasassessed in normal human serum (n=96) using the ProteOn XPR36 (Bio-RadLaboratories, Inc.). PBS/Tween (phosphate buffered saline, pH7.4, 0.005%Tween20) was used as running buffer and the experiments were performedat 25° C.

ISVDs are captured on the chip via binding of the ALB23002 buildingblock to HSA, which is immobilized on the chip. To immobilize HSA, theligand lanes of a ProteOn GLC Sensor Chip are activated with EDC/NHS(flow rate 30 μl/min) and HSA is injected at 100 μl/ml in ProteOnAcetate buffer pH4.5 to render immobilization levels of approximately2900 RU. After immobilization, surfaces are deactivated withethanolamine HCl (flow rate 34 μl/min).

Subsequently, ISVD constructs are injected for 2 min at 45 μl/min overthe HSA surface to render an ISVD capture level of approximately 800 RU.The samples containing pre-existing antibodies are centrifuged for 2minutes at 14,000 rpm and supernatant is diluted 1:10 in PBS-Tween20(0.005%) before being injected for 2 minutes at 45 μl/min followed by asubsequent 400 seconds dissociation step. After each cycle (i.e. beforea new ISVD capture and blood sample injection step) the HSA surfaces areregenerated with a 2 minute injection of HCl (100 mM) at 45 μl/min.Sensorgrams showing pre-existing antibody binding are obtained afterdouble referencing by subtracting 1) ISVD-HSA dissociation and 2)non-specific binding to reference ligand lane. Binding levels ofpre-existing antibodies are determined by setting report points at 125seconds (5 seconds after end of association). Percentage reduction inpre-existing antibody binding is calculated relative to the bindinglevels at 125 seconds of a reference ISVD.

The pentavalent ISVD construct F027400161, optimized for reducedpre-existing antibody binding by introduction of mutations L11V and V89Lin each building block and a C-terminal alanine, shows substantiallyless binding to pre-existing antibodies compared to a controlnon-optimized pentavalent ISVD construct F027301186, (Table 24 and FIG.5).

Pre-existing antibody binding depends on the orientation of the buildingblocks and the linker lengths present in the multispecific constructs.Table 24 and FIG. 5 demonstrate that construct F027400161 shows lowerpre-existing antibody reactivity than construct F027400163, due to itsspecific orientation, but that F027400164 shows lower reactivity thanF027400161, due to short linkers all over.

6.4.8 Example 23: F027400161 Blocks TSLP-Induced CCL17 in HumanDendritic Cells In Vitro

The type 2 inflammation cascade is initiated and propagated by aconcerted action of epithelial cells, dendritic cells, type 2 helper Tcells (Th2 cells), mast cells and innate lymphoid cells in acontext-dependent manner. The cytokine thymic stromal lymphopoietin(TSLP) has been implicated in the initiation and progression of allergicinflammation through its ability to activate dendritic cells (DCs). Uponactivation by TSLP, human DCs produce CCL17, a Th2-associated chemokine,and drive Th2 cell differentiation from naïve CD4⁺ T cells. F027400161targets both TSLP and IL-13, can block the interaction between TSLP andDCs, and thereby, reduce CCL-17 production and is projected to conferefficacy in type 2 inflammatory diseases and beyond.

The ability of F027400161 to inhibit TSLP-induced CCL17 production wasassessed in human DCs isolated from 8 individual healthy donors incomparison to the reference monospecific antibody, anti-hTSLP referencemAb1. Human DCs (CD3⁻CD14⁻CD11c⁺HLA-DR^(high)) were isolated andenriched from healthy human PBMCs in buffy coat (human leucocyte pack)samples. A total of 0.5-0.8×10⁶ DCs/well were incubated with eight3-fold serially diluted concentrations of F027400161 (400 ng/mL or 5.714nM top concentration) or eight 4-fold serially diluted concentrations ofanti-hTSLP reference mAb1 (4000 ng/mL or 27 nM top concentration) priorto stimulation with 4 ng/mL of recombinant human TSLP for 36 hours in37° C. cell culture incubator. Recombinant human TSLP is commerciallyavailable, such as from R&D Systems (Catalog number 1398-TS). CCL17production in freshly collected cell culture supernatant was measured byELISA and IC₅₀ values of the ISVD construct and benchmark antibody werecalculated in Graphpad Prism.

The collective results of the dose inhibition responses of F027400161and anti-hTSLP reference mAb1 in human DCs, are shown in FIG. 6. Thereference monospecific antibody anti-hTSLP reference mAb1 inhibited TSLPinduced CCL17 production at a mean IC₅₀ concentration of 793.4 pM, whileF027400161 inhibited TSLP induced CCL17 production with a mean IC₅₀ of53.26 pM.

In conclusion, these results demonstrate that the ISVD constructF027400161 is more potent in inhibiting TSLP-induced CCL17 response inhuman DCs, compared to anti-hTSLP reference mAb1.

6.4.9 Example 24: F027400161 Blocks 0.5 ng/mL [IL-13+TSLP] InducedSynergistic CCL17 in Human PBMCs In Vitro

Type 2 cytokines such as thymic stromal lymphopoietin (TSLP) andInterleukin-13 (IL-13) exert unique, additive and synergistic responseto drive asthma and atopic dermatitis (AD) pathophysiology. The roles ofTSLP as an epithelial cell-derived initiator of the type 2 immunecascade and of IL-13 as a downstream effector cytokine have beenextensively validated. F027400161 targets both TSLP and IL-13, therebyprojected to confer efficacy in type 2 mediated inflammatory diseasesand beyond.

The ability of F027400161 to inhibit 0.5 ng/mL IL-13 and TSLP-inducedsynergistic production of CCL17 (TARC) was evaluated in human PBMCs from8 individual healthy donors. The study was designed to evaluatenon-inferiority of the ISVD construct versus monospecific biologics,anti-hTSLP reference mAb1 and anti-hIL-13 reference mAb1. One millioncells per well of human PBMCs were stimulated with 0.5 ng/mL ofrecombinant human TSLP (recombinant human TSLP is commerciallyavailable, such as from R&D Systems (cat nr 1398-TS) plus IL-13 (R&DSystems, cat nr 213-ILB-005/CF) and incubated with ten 3-fold seriallydiluted concentrations of the ISVD construct (10 nM top concentration),anti-hIL-13 reference mAb1 (10 nM top concentration) and anti-hTSLPreference mAb1 (100 nM top concentration) in a 96-well plate for 20hours in 37° C. cell-culture incubator. The assays were performed withtechnical triplicates within each donor for F027400161. Theconcentrations of the cytokines used were within 2-fold of the standarderror of reported literature values from sera, BAL fluid, sputum andskin of normal humans, asthmatics and atopic dermatitis patients(Berraïes A et al, Immunol Letter 178: 85-91, 2016; Bellini A et al,Mucosal Immunology 5(2):140-9, 2012; Davoodi P et al, Cytokine60(2):431-7, 2012; Szegedi K et al, J Eur Acad Dermatol Venereol29(11):2136-44, 2015). CCL17 production in freshly collected cellculture supernatant was measured by Meso Scale Diagnostics (MSD) V-PLEXHuman TARC Kit.

The collective results of the dose inhibition responses of F027400161and benchmark antibodies, anti-hIL-13 reference mAb1 and anti-hTSLPreference mAb1, are shown in FIG. 7. F027400161 demonstrated 100%inhibition of 0.5 ng/mL IL-13+TSLP-induced synergistic CCL17 productionwith a mean IC₅₀ of 0.0061 nM. The reference antibody anti-hIL-13 mAb1,despite having a lower mean IC₅₀ of 0.0028 nM, was unable to fully blockthe synergistic CCL17 response at equimolar doses of F027400161 andplateaued at approximately 80% inhibition. On the other hand, anti-hTSLPreference mAb1 was only able to block approximately 50% of the CCL17production with a mean IC₅₀ of 2.932 nM.

In conclusion, these results demonstrate that F027400161 is more potentthan anti-hTSLP reference mAb1 and is superior compared to anti-hIL-13reference mAb1 for blocking pathophysiological relevant concentrationsof IL-13 and TSLP-induced synergistic response in human PBMCs,highlighting its therapeutic potential for the treatment of type 2inflammatory diseases such as asthma and atopic dermatitis.

6.4.10 Example 25: F027400161 Blocks 5 ng/mL [IL-13+TSLP] InducedSynergistic CCL17 in Human PBMCs In Vitro

The Ability of F027400161 to Inhibit 5 ng/mL IL-13+TSLP-Induced CCL17production was evaluated in human PBMCs from 8 healthy individualdonors. The study was designed to evaluate non-inferiority of the ISVDconstruct versus monospecific biologics, anti-hTSLP reference mAb1 andanti-IL-13 reference mAb1. The concentrations of the cytokines used were^(˜)10 fold over the upper end of the pathophysiological ranges of TSLPand IL-13 that have been reported in the literature in normal humans,asthmatics and atopic dermatitis patients (Berraïes A et al, ImmunolLetter 178: 85-91, 2016; Bellini A et al, Mucosal Immunology 5(2):140-9,2012; Davoodi P et al, Cytokine 60(2):431-7, 2012; Szegedi K et al, JEur Acad Dermatol Venereol 29(11):2136-44, 2015) as hypotheticalconcentrations during transient inflammatory state. One million cellsper well of human PBMCs were stimulated with 5 ng/mL of recombinanthuman TSLP plus IL-13 (R&D Systems, cat nr 213-ILB-005/CF) and incubatedwith ten 3-fold serially diluted concentrations of F-27400161 (10 nM topconcentrations), anti-hIL-13 reference mAb1 (10 nM top concentration)and anti-hTSLP reference mAb1 (100 nM top dose) in a 96-well plate for20 hours in 37° C. cell-culture incubator. The assays were performedwith technical triplicates within each donor for F027400161. CCL17production in freshly collected cell culture supernatant was measured byMeso Scale Diagnostics (MSD) V-PLEX Human TARC Kit.

The collective results of the dose inhibition responses of F027400161and benchmark antibodies, anti-hIL-13 reference mAb1 and anti-hTSLPreference mAb1, are shown in FIG. 8. F027400161 demonstrated 100%inhibition of 5 ng/mL IL-13+TSLP-induced synergistic CCL17 productionwith a mean IC₅₀ of 0.0387 nM. While PBMCs treated with equimolar dosesof the comparator antibody anti-hIL-13 reference mAb1 showed a lowermean IC₅₀ of 0.01339 nM, the inhibition response never reached 100% andplateaued at approximately 90% inhibition. On the other hand, anti-hTSLPreference mAb1 only partially blocked approximately 40% of the CCL17production with a mean IC₅₀ of 19 nM.

In conclusion, these results demonstrate that F027400161 is superior toboth anti-hTSLP reference mAb1 and anti-hIL-13 reference mAb1 inblocking IL-13 and TSLP-induced synergistic CCL17 response in humanPBMCs at [TSLP+IL-13] concentrations 10-fold over the pathophysiologicalranges of the cytokines, highlighting its therapeutic potential for thetreatment of asthma and atopic dermatitis during acute or inflammatoryphases.

6.4.11 Experiment 27: T027400161 Blocks Allergen Der P-InducedProduction of IL-5, CCL17, and CCL26 in a Triculture Assay System

TSLP drives type 2 immune response by inducing CCL17, IL-5, and IL-13production. Subsequently, IL-13 triggers CCL26 production by localepithelial cells, leading to the ramification of type 2 immune responsemediated inflammatory diseases and beyond.

The ability of F027400161 to inhibit TSLP-induced IL-5 and CCL17, andIL-13-induced CCL26 production was evaluated in a triculture assaysystem using MRCS fibroblasts and A549 epithelial cells coculturing withDer P-stimulated human PBMCs from 6 individual normal donors for 6 days.The study was designed to evaluate non-inferiority of the ISVD versusmonospecific biologics, anti-hTSLP reference mAb1 and anti-hIL-13reference mAb1. MRCS fibroblasts produced ^(˜)100 pg/mL endogenous TSLPconstitutively, and A549 epithelial cells produced CCL26 in response toIL-13 produced by PBMCs with Der P and endogenous TSLP stimulation. Oneday prior to coculture with human PBMCs, seventy-five thousand MRC5fibroblasts and A549 epithelial cells per well were plated. One millioncells per well of human PBMCs were added into plated MRC5 fibroblastsand A549 epithelial cells, stimulated with 3 μg/mL of Der P, andincubated with 11.1 nM of ISVD, anti-hIL-13 reference mAb1, oranti-hTSLP reference mAb1 in a 24-well plate for 6 days in a 37° C.cell-culture incubator. The assays were performed with technicaltriplicates within each donor for F027400161. The production of IL-5,CCL17, and CCL26 in freshly collected cell culture supernatant wasmeasured by Human Magnetic Luminex Assays from RnD System.

The collective results of the inhibition responses of F027400161 andbenchmark antibodies, anti-hIL-13 reference mAb1 and anti-hTSLPreference mAb1, are shown in FIG. 9. F027400161 demonstrated 60%inhibition of CCL17, 50% inhibition of IL-5, and 95% inhibition of CCL26production. The reference antibody anti-hTSLP reference mAb1 displayed50% inhibition of CCL17, 50% inhibition of IL-5, and approximately 55%inhibition of CCL26 production. While anti-hIL-13 reference mAb1demonstrate comparable 95% inhibition of CCL26 production, thisreference antibody was only able to block approximately 35% of the CCL17production and less than 10% of IL-5 production (FIG. 9). Lack ofcomplete inhibition of IL-5 and CCL17 by these tested molecules maysuggest that Der P stimulation triggers PBMCs to elicit pathways otherthan TSLP and IL-13 to drive the production of IL-5 and CCL17.

In conclusion, these results demonstrate that the anti-TSLP/IL-13 ISVDF027400161 is superior than anti-hTSLP reference mAb1 and anti-hIL-13reference mAb1 by its ability to block three cytokines and chemokines(CCL17, IL-5 and CCL26) in a complex assay system comprising of humanPBMCs cocultured with tissue structural cells, highlighting itstherapeutic potential for the treatment of type 2 inflammatory diseasessuch as asthma and atopic dermatitis, as well as a broad range ofimmunological disease indications.

6.4.12 Example 26: NSG-SGM3 Mouse Model to Evaluate F027400161 MediatedTarget Occupancy and Pharmacodynamics In Vivo

F027400161 targets both human TSLP and IL-13 and does not cross reactwith the murine orthologs. Hence, to evaluate the biological activitiesof F027400161, a xenografted, humanized model system was used. FemaleNSG-SGM3 (NOD/SCID-IL2Rγ−/−, NOD.Cg-Prkdcscidll2rytm1Wjl/SzJ) wereobtained from Jackson Labs, Bar Harbor, Me., USA. These mice expresshuman hematopoietic cytokines: stem cell factor (SCF),granulocyte/macrophage stimulating factor (GM-CSF), and interleukin-3(IL-3), all driven by a human cytomegalovirus promoter/enhancersequence. The triple transgenic mouse produces above cytokinesconstitutively, providing cell proliferation and survival signals,supporting the stable engraftment of CD33+ myeloid lineages, and severaltypes of lymphoid cells. Briefly, the protocol followed for engraftmentis as follows:

On day 0 of the study, mice were irradiated with 150 centiGray at a rateof 120 rads/minute for 1 minute and 15 seconds. Mice were engrafted with1×10⁵ cord blood CD34+ stem/progenitor cells by the intravenous (IV)route in 200 μl of Dulbecco's phosphate buffered saline (DPBS)approximately 6 hours post-engraftment. One group of mice wereirradiated in the same manner, but not engrafted. These mice areconsidered irradiated naïve mice. On day 88 post engraftment, micereceived a hydrodynamic (HDD) i.v. injection of either saline (engraftedcontrol mice) or 50 μg of IL-4 minicircle DNA in combination with 50 μgof TSLP minicircle DNA. On day 91, a submandibular bleed was performed,and 100 to 150 μl of blood was collected from each mouse and placed intolithium heparin tubes. An engraftment check was performed by flowcytometry and the plasma levels of IL-4 and TSLP were evaluated.Information from the engraftment check and/or the cytokine determinationwas used to select (assign or eliminate) mice from the study. Mice fromthe engrafted control group with less than 25% human CD45+ cells wereremoved from the study. Similarly, engrafted mice that receivedminicircle DNA and showed plasma TSLP levels 1 standard deviation belowthe mean were also removed from the study. Mice that received minicircleDNA by HDD i.v. injection received subcutaneous doses of either vehicle(20 mM phosphate, 125 mM L-arginine HCL, and 0.01% tween 20 pH 7.0) orF027400161 (0.01, 0.05, 0.1, or 10 mg/kg) on days 95, 97, 99, and 101.On day 103, mice were anesthetized by isoflurane anesthesia. While underisoflurane anesthesia, blood was collected by retro-orbital bleeds.Following blood collection and while still under isoflurane anesthesia,the mice were terminated by cervical dislocation. A portion of the lungwas harvested and placed in RNA later for gene expression evaluation.Plasma levels of human TSLP from the day 103 plasma samples weredetermined by MSD kit evaluation (Cat #K15067-L-2, Meso ScaleDiagnostics, Rockville, Md., USA). Plasma levels of human IL-13 from theday 103 plasma samples were determined by ELISA (Cat #88-7439-88HumanIL-13 ELISA kit, Invitrogen/Thermo-Fischer, Waltham, Mass., USA).Internal assay validation experiments demonstrated that both the humanTSLP and IL-13 detection kits were not able to detect F027400161 boundhTSLP and hIL-13.

The collective results of these experiments as shown in FIG. 10 and FIG.11 demonstrate that F027400161 was able to significantly inhibitdetectable levels of human TSLP and IL-13 in the plasma of humanizedNSG-SGM3 mice, demonstrating target occupancy for both human TSLP andhuman IL-13.

In the NSG-SGM3 mouse model, hydrodynamic delivery of TSLP and IL-4cDNAs turn on the expression of human IL-13 (FIG. 11). Pharmacodynamiceffects of F027400161 were studied using samples derived from theNSG-SGM3 study by taking advantage of the ability of human IL-13 tosignal through the mouse IL-13 receptor (Hershey GK. 2003, J AllergyClin Immunol.; 111(4):677-90). In these studies, the impact ofF027400161 treatment on human IL-13 regulated mouse gene transcriptswere studied. Mouse lung tissues from the above study were used in thisanalysis.

The lungs from treated and control NSG-SGM3 mice were harvested andprocessed to make RNA as detailed in the attached protocol. The RNAswere processed for quantification by TaqMan and the data analyzed asdescribed in the protocols. In brief, lungs harvested from the mice werestored in RNALater, processed and purified according to standardprotocols to generate high quality RNA. Purified lung RNA was thenreverse transcribed to cDNA using Quanta 0-Script 5× master mixaccording to manufacturer's protocol. Obtained lung cDNA was used toquantify the transcript expression levels of the human IL-13 responsivemouse target genes (mouse Retnla and mouse Clca1) and an endogenouscontrol (Rpl37a) using a TaqMan assay according to manufacturer'sprotocols. Data analysis was performed in Quantstudio 6&7 flex software.For each probe, C_(T) values and delta C_(T) values (against Rpl37a)were exported into excel and relative expression values for each genewere calculated using the following formula:

Normalized relative expression=(Power(2,−(delta CT)))*1000.

The two human IL-13 regulated mouse genes evaluated were Retnla(Resistin like alpha), that plays a role in pulmonary vascularremodeling and Clca1 (chloride channel accessory 1) (Lewis CC, 2009, JAllergy Clin Immunol.; 123(4):795-804).

The collective results of these experiments as shown in FIGS. 12 and 13demonstrate that F027400161 was able to significantly inhibit mouseRetnla and mouse Clca1 transcript expression, demonstrating the in vivopharmacodynamics effect of F027400161 on human IL-13 driven mousetranscript responses.

Methods:

Sample Homogenate Preparation:

Lungs were harvested from the mice and stored in RNA later. Lung lobeswere then dried and transferred to fastprep lysing Matrix A tubes (forhomogenizing lungs) containing 1 mL RLT+2-ME. Samples were homogenizedin MP-Bio homogenizer using program 1 (Two 40 second cycles separated by5 minutes to avoid sample heatup). Samples were then spun at 10,000 gfor 3 minutes. Collected 350 ul of lysate [in RLT+2ME (1% v/v)]. Pipetteusing a multi-channel multiple times (^(˜)20×) to lyse the cells.

RNA-Preparation:

For RNA purification, 350 ul homogenate was used. 1× volume (350 ul) of70% Ethanol was added and the homogenate mixed thoroughly andtransferred to a 96 well RNeasy spin plate placed in elution plate andRNA was prepared using the Qiagen RNA mini tissue RNA extractionprotocol with following modifications. The 96 well plate was coveredwith sealable aluminum foil and centrifuged for 2 minutes at 4000×g. Towash the column, 400 μl Buffer RWT was added to the RNeasy spin columnsand the spin-column plate was centrifuged for 2 minutes at RT at 4000×g.DNAse I digestion was carried out by adding 80 ul of 1×DNAse I mix andincubating at RT for 15 minutes. The DNAse I was washed off by adding400 ul buffer RWT and spinning the plate at 4000×g for 2 minutes. Thiswas followed by washing the spin-plate with 500 ul each of buffer RPEand 80% ethanol. This was followed by drying of column membranes byspinning at 4000×g for 4 minutes. RNA was then eluted in 40 μl Tris HCl(10 mM; pH-8.0). RNA was quantified using Nanodrop and 500 ng RNA wasused for cDNA preparation.

First Strand Synthesis:

Quanta 0-Script 5× master mix was used and cDNA was synthesized usingmanufacturer's protocol. Final concentration of cDNA was 25 ng/ul. cDNAwas stored at −20 Celsius till the TaqMan assay was performed.

TaqMan Assay:

TaqMan multiplex master mix was prepared in a 1.5 ml microcentrifugetube by adding the components in the following order. Three separatemaster mixes made for each probe-set along with the internal Rpl37acontrol. Each multiplex qPCR reaction was conducted in a 10 μl reactionvolume.

Component Single Rxn (ul) For 40 Rxns (ul) 50X RPL37A Primer/Probe 0.310 20X Target Gene Primer/Probe 0.6 24 Water 2.8 4 TaqMan Fast AdvancedMastermix; 2X 5.0 200 Total 8.7 238

A total of 8.8 ul of the master mix was added for each sample into theappropriate wells of a 384-well optical plate. 30 ng (1.2 ul) cDNAsamples were added to each well. TaqMan assay was set-up on QuantStudio7K. The conditions in the thermo cycler were: pre-denaturation at 95° C.for 3 min, 40 cycles of denaturation at 95° C. for 2 s, and annealingand extension at 60° C. for 5 s. Fluorescent measurements were carriedout during the extension step.

Data Analysis:

Data analysis was performed in Quantstudio 6&7 flex software.

For each probe, CT values and delta CT values (against Rpl37a) wereexported into excel and relative expression values for each gene werecalculated using the following formula:

Normalized relative expression=(power(2,−(delta CT)))*1000

REFERENCES

-   Liu, Y. J. 2006, J Exp Med.; 203(2):269-73. Thymic stromal    lymphopoietin: master switch for allergic inflammation.-   Hershey G K. 2003, J Allergy Clin Immunol.; 111(4):677-90. IL-13    receptors and signaling pathways: an evolving web.-   Lewis C C, Aronow B, Hutton J, Santeliz J, Dienger K, Herman N,    Finkelman F D, Wills-Karp M. 2009, J Allergy Clin Immunol.;    123(4):795-804. Unique and overlapping gene expression patterns    driven by IL-4 and IL-13 in the mouse lung.

7. INDUSTRIAL APPLICABILITY

The polypeptides, nucleic acid molecules encoding the same, vectorscomprising the nucleic acids and compositions described herein may beused for example in the treatment of subjects suffering frominflammatory diseases.

1. A method of treating an inflammatory disease, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of a polypeptide that comprises at least one immunoglobulin single variable domain (ISVD) that specifically binds to IL13 or TSLP, wherein said ISVD comprises three complementarity determining regions (CDR1 to CDR3, respectively); and wherein the at least one ISVD comprises: a) a CDR1 that is the amino acid sequence of SEQ ID NO: 7; a CDR2 that is the amino acid sequence of SEQ ID NO: 12; and a CDR3 that is the amino acid sequence of SEQ ID NO: 17, b) a CDR1 that is the amino acid sequence of SEQ ID NO: 8, a CDR2 that is the amino acid sequence of SEQ ID NO: 13; and a CDR3 that is the amino acid sequence of SEQ ID NO: 18, c) a CDR1 that is the amino acid sequence of SEQ ID NO: 9; a CDR2 that is the amino acid sequence of SEQ ID NO: 14; and a CDR3 that is the amino acid sequence of SEQ ID NO: 19, or d) a CDR1 that is the amino acid sequence of SEQ ID NO: 11; a CDR2 that is the amino acid sequence of SEQ ID NO: 16; and a CDR3 that is the amino acid sequence of SEQ ID NO:
 21. 2.-3. (canceled)
 4. The method according to claim 1, wherein said at least one ISVD that specifically binds to IL-13 or TSLP comprises: a) the amino acid sequence of SEQ ID NO: 2, b) the amino acid sequence of SEQ ID NO: 3, c) the amino acid sequence of SEQ ID NO: 4, or d) the amino acid sequence of SEQ ID NO:
 6. 5. The method according to claim 1, wherein the polypeptide comprises at least two ISVDs, wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least two ISVDs are optionally linked via one or more peptidic linkers, and wherein: a) a first and a second ISVD specifically binds to IL-13 and comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17, b) a first and a second ISVD specifically binds to IL-13 and comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 8; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 13; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18, c) a first ISVD specifically binds to IL-13 and comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17, and  a second ISVD specifically binds to IL-13 and comprises iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 8; v. a CDR2 that is the amino acid sequence of SEQ ID NO: 13; and vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 18, d) a first ISVD specifically binds to IL-13 and comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 8; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 13; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 18, and  a second ISVD specifically binds to IL-13 and comprises iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 7; v. a CDR2 that is the amino acid sequence of SEQ ID NO: 12; and vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 17, e) a first ISVD specifically binds to TSLP and comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 11; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 16; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21, and  a second ISVD specifically binds to TSLP and comprises iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 9; v. a CDR2 that is the amino acid sequence of SEQ ID NO: 14; and vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 19, or f) a first ISVD specifically binds to TSLP and comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 9; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 14; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 19, and  a second ISVD specifically binds to TSLP and comprises iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 11; v. a CDR2 that is the amino acid sequence of SEQ ID NO: 16; and vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 21, wherein the order of the ISVDs indicates their relative position to each other considered from the N-terminus to the C-terminus of said polypeptide.
 6. The method according to claim 5, wherein the polypeptide comprises: a) the amino acid sequence of SEQ ID NO: 148, b) the amino acid sequence of SEQ ID NO: 149, c) the amino acid sequence of SEQ ID NO: 150, d) the amino acid sequence of SEQ ID NO: 151, e) the amino acid sequence of SEQ ID NO: 152, f) the amino acid sequence of SEQ ID NO: 153, g) the amino acid sequence of SEQ ID NO: 154, h) the amino acid sequence of SEQ ID NO: 155, i) the amino acid sequence of SEQ ID NO: 156, j) the amino acid sequence of SEQ ID NO: 157, k) the amino acid sequence of SEQ ID NO: 158, or l) the amino acid sequence of SEQ ID NO:
 159. 7. The method according to claim 1, wherein the polypeptide comprises at least four ISVDs, wherein each of said ISVDs comprises three complementarity determining regions (CDR1 to CDR3, respectively), wherein the at least four ISVDs are optionally linked via one or more peptidic linkers, and wherein: a) a first ISVD specifically binds to IL-13 and comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 7; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 12; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 17, b) a second ISVD specifically binds to IL-13 and comprises iv. a CDR1 that is the amino acid sequence of SEQ ID NO: 8; v. a CDR2 that is the amino acid sequence of SEQ ID NO: 13; and vi. a CDR3 that is the amino acid sequence of SEQ ID NO: 18, c) a third ISVD specifically binds to TSLP and comprises vii. a CDR1 that is the amino acid sequence of SEQ ID NO: 9; viii. a CDR2 that is the amino acid sequence of SEQ ID NO: 14; and ix. a CDR3 that is the amino acid sequence of SEQ ID NO: 19, and d) a fourth ISVD specifically binds to TSLP and comprises x. a CDR1 that is the amino acid sequence of SEQ ID NO: 11; xi. a CDR2 that is the amino acid sequence of SEQ ID NO: 16; and xii. a CDR3 that is the amino acid sequence of SEQ ID NO: 21, wherein the order of the ISVDs indicates their relative position to each other considered from the N-terminus to the C-terminus of said polypeptide.
 8. (canceled)
 9. The method according to claim 7, wherein: a) said first ISVD comprises the amino acid sequence of SEQ ID NO: 2; b) said second ISVD comprises the amino acid sequence of SEQ ID NO: 3; c) said third ISVD comprises the amino acid sequence of SEQ ID NO: 4; and d) said fourth ISVD comprises the amino acid sequence of SEQ ID NO:
 6. 10. The method according to claim 1, wherein said polypeptide further comprises one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.
 11. The method according to claim 10, in which said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is chosen from the group consisting of binding units that can bind to serum albumin (such as human serum albumin) or a serum immunoglobulin (such as IgG).
 12. The method according to claim 11, in which said binding unit that provides the polypeptide with increased half-life is an ISVD that can bind to human serum albumin.
 13. The method according to claim 12, wherein said ISVD binding to human serum albumin comprises the amino acid sequence of SEQ ID NO:
 5. 14. The method according to claim 7, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO:
 1. 15.-18. (canceled)
 19. The method according to claim 1, wherein the inflammatory disease is a type 2 inflammatory disease.
 20. The method according to claim 19, wherein the type 2 inflammatory disease is atopic dermatitis.
 21. The method according to claim 12, wherein the ISVD binding to human serum albumin comprises i. a CDR1 that is the amino acid sequence of SEQ ID NO: 10 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 10; ii. a CDR2 that is the amino acid sequence of SEQ ID NO: 15 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 15; and iii. a CDR3 that is the amino acid sequence of SEQ ID NO: 20 or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO:
 20. 22. The method according to claim 12, wherein the ISVD binding to human serum albumin comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 10, a CDR2 that is the amino acid sequence of SEQ ID NO: 15 and a CDR3 that is the amino acid sequence of SEQ ID NO:
 20. 23. The method according to claim 12, wherein the amino acid sequence of said ISVD binding to human serum albumin comprises a sequence identity of more than 90% with SEQ ID NO:
 5. 24. The method according to claim 14, wherein the polypeptide consists of the amino acid sequence of SEQ ID NO:
 1. 25. The method according to claim 1, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of a composition comprising said polypeptide. 